Bacterial efflux pump inhibitors

ABSTRACT

Disclosed herein are compounds of formula I and salts thereof. Also disclosed are compositions comprising compounds of formula I and methods using compounds of formula I.

RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.62/511,851 that was filed on May 26, 2017 and to U.S. ProvisionalApplication No. 62/523,156 that was filed on Jun. 21, 2017. The entirecontent of each of these applications is hereby incorporated byreference herein.

BACKGROUND OF THE INVENTION

Antibiotics have been effective tools in the treatment of infectiousdiseases. However, bacteria have developed several different mechanismsto overcome the action of antibiotics. These mechanisms of resistancecan be specific such as for a molecule or a family of antibiotics, orthe mechanisms can be non-specific. Several mechanisms of resistance canexist in a single bacterial strain, and those mechanisms may actindependently or they may act synergistically to overcome the action ofan antibiotic or a combination of antibiotics. Specific mechanismsinclude, for example, degradation of the drug, inactivation of the drugby enzymatic modification, and alteration of the drug target. Additionalmechanisms of drug resistance include mechanisms in which access of theantibiotic to the target is prevented or reduced by decreasing thetransport of the antibiotic into the cell or by increasing the efflux ofthe drug from the cell to the outside medium. Both of these mechanismscan lower the concentration of drug at the target site and allowbacterial survival in the presence of one or more antibiotics that wouldotherwise inhibit or kill the bacterial cells. Some bacteria utilizeboth mechanisms, combining low permeability of the cell wall (includingmembranes) with an active efflux of antibiotics. It has been shown thatefflux of antibiotics can be mediated by more than one pump in a singleorganism and that almost all antibiotics are subject to resistance bythis mechanism.

These multiple resistance mechanisms have become widespread and threatenthe clinical utility of antibacterial therapy. The increase inantibiotic resistant strains has been particularly noted in majorhospitals and care centers. The consequences of the increase inresistant strains include, for example higher morbidity and mortality,longer patient hospitalization, and an increase in treatment costs.Accordingly, there is a need for agents and methods for inhibiting oneor more of these mechanisms of bacterial resistance.

SUMMARY OF THE INVENTION

Compounds disclose herein, when tested in combination with a knownantibiotic, lower the minimum inhibitory concentration of the knownantibiotic to inhibit bacterial cell growth.

Not to be bound by theory the compounds are believed to exert thiseffect by the inhibition of a bacterial efflux pump(s).

Accordingly, one embodiment provides a compound of formula I:

wherein:

A is —C(═O)N(R^(a1))—R¹, —(C₁-C₃)alkyl-C(═O)N(R^(a1))R¹,—(C₁-C₃)alkyl-O—R¹, —O—R¹, —(C₁-C₃)alkyl-N(R^(a1))—R¹, —N(R^(a1))—R¹, orR¹;

B is (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy,(C₁-C₄)haloalkoxy, (C₃-C₇)carbocyclyl, (C₃-C₇)carbocyclyl-(C₁-C₄)alkyl-,(C₃-C₇)carbocyclyl-(C₂-C₆)alkynyl-, aryl, aryl-(C₁-C₄)alkyl-,heteroaryl, heteroaryl-(C₁-C₄)alkyl-, 3-7membered-monocyclic-heterocycle, or 3-7membered-monocyclic-heterocycle-(C₁-C₄)alkyl- wherein any(C₃-C₇)carbocyclyl, (C₃-C₇)carbocyclyl-(C₁-C₄)alkyl-,(C₃-C₇)carbocyclyl-(C₂-C₆)alkynyl-, aryl, aryl-(C₁-C₄)alkyl-,heteroaryl, heteroaryl-(C₁-C₄)alkyl-, 3-7membered-monocyclic-heterocycle, or 3-7membered-monocyclic-heterocycle-(C₁-C₄)alkyl- of B is optionallysubstituted with one or more Z¹ groups;

each R¹ is independently:

(a) (C₁-C₁₄)alkyl substituted with one or more groups selected from thegroup consisting of —NR^(b2)R^(c2), —NHNH₂, —C(═NR^(a2))(NR^(b2)R^(c2)),—NR^(a2)C(═NR^(a2))(R^(d2)), and —NR^(a2)C(═NR^(a2))(NR^(b2)R^(c2)) andwherein (C₁-C₁₄)alkyl is optionally substituted independently with oneor more halo, (C₁-C₄)alkyl or (C₃-C₇)carbocyclyl; or

(b) (C₃-C₇)carbocyclyl, (C₃-C₇)carbocyclyl-(C₁-C₄)alkyl-, 4-7 memberedmonocyclic heterocyclyl, or 4-7 membered monocyclicheterocyclyl-(C₁-C₄)alkyl-, wherein each (C₃-C₇)carbocyclyl or(C₃-C₇)carbocyclyl-(C₁-C₄)alkyl- is independently substituted with oneor more Z² or Z³, and wherein each 4-7 membered monocyclic heterocyclylor 4-7 membered monocyclic heterocyclyl-(C₁-C₄)alkyl- is independentlyoptionally substituted with one or more Z² or Z³, and wherein any(C₃-C₇)carbocyclyl, (C₃-C₇)carbocyclyl-(C₁-C₄)alkyl-, 4-7 memberedmonocyclic heterocyclyl, or 4-7 membered monocyclicheterocyclyl-(C₁-C₄)alkyl- of R¹ is optionally substituted independentlywith one or more halo, (C₁-C₄)alkyl or (C₃-C₇)carbocyclyl; R² ishydrogen, (C₁-C₄)alkyl or phenyl(C₁-C₃)alkyl-, wherein the phenyl isoptionally substituted with one or more (C₁-C₄)alkyl, —O(C₁-C₄)alkyl,halogen, or —NO₂;

R³ is hydrogen, halo, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy,(C₁-C₄)haloalkoxy, aryl, —OH, or heteroaryl wherein the aryl orheteroaryl is optionally substituted with one or more groupsindependently selected from the group consisting of halo, —OH, —NO₂,—CN, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, and(C₁-C₄)haloalkoxy;

R⁴ is hydrogen, halo, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy,(C₁-C₄)haloalkoxy, aryl, —OH, or heteroaryl wherein the aryl orheteroaryl is optionally substituted with one or more groupsindependently selected from the group consisting of halo, —OH, —NO₂,—CN, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, and(C₁-C₄)haloalkoxy;

R⁵ is hydrogen, halo, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy,(C₁-C₄)haloalkoxy, aryl, —OH, or heteroaryl wherein the aryl orheteroaryl is optionally substituted with one or more groupsindependently selected from the group consisting of halo, —OH, —NO₂,—CN, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, and(C₁-C₄)haloalkoxy;

R⁶ is hydrogen, halo, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy,(C₁-C₄)haloalkoxy, aryl, —OH, or heteroaryl wherein the aryl orheteroaryl is optionally substituted with one or more groupsindependently selected from the group consisting of halo, —OH, —NO₂,—CN, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, and(C₁-C₄)haloalkoxy;

each Z¹ is independently halo, —OH, —NO₂, —CN, (C₁-C₄)alkyl,(C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, —CO₂H, CO₂H, benzyloxy (—OCH₂-phenyl),or (C₁-C₄)haloalkoxy;

each Z² is independently selected from the group consisting of—NR^(b3)R^(c3), —NHNH₂, —C(═NR^(a3))(NR^(b3)R^(c3)),—NR^(a3)C(═NR^(a3))(R^(d3)), and —NR^(a3)C(═NR^(a3))(NR^(b3)R^(c3)) eachZ³ is independently —(C₁-C₆)alkyl substituted with one or more Z² andoptionally optionally substituted with one or more Z⁴;

each Z⁴ is independently halo or (C₃-C₇)carbocyclyl;

each R^(a1) is independently hydrogen, (C₁-C₄)alkyl or(C₃-C₇)carbocyclyl;

each R^(a2) is independently hydrogen, (C₁-C₄)alkyl or(C₃-C₇)carbocyclyl;

each R^(b2) and R² is independently hydrogen, (C₁-C₄)alkyl or(C₃-C₇)carbocyclyl;

R^(d2) is (C₁-C₄)alkyl or (C₃-C₇)carbocyclyl;

each R^(a3) is independently hydrogen (C₁-C₄)alkyl or(C₃-C₇)carbocyclyl;

each R^(b3) and R³ is independently hydrogen (C₁-C₄)alkyl or(C₃-C₇)carbocyclyl; and

R^(d3) is (C₁-C₄)alkyl or (C₃-C₇)carbocyclyl;

or a salt thereof.

One embodiment provides a pharmaceutical composition comprising acompound of formula I or a pharmaceutically acceptable salt thereof asdescribed herein, and a pharmaceutically acceptable vehicle.

One embodiment provides pharmaceutical composition comprising a compoundof formula I or a pharmaceutically acceptable salt thereof as describedherein, one or more antibacterial agents and a pharmaceuticallyacceptable vehicle.

One embodiment provides a method of inhibiting a bacterial efflux pumpin an animal (e.g., a mammal such as a human) comprising administeringto the animal a compound of formula I or a pharmaceutically acceptablesalt thereof as described herein.

One embodiment provides a method of inhibiting a bacterial efflux pumpin an animal (e.g., a mammal such as a human) comprising administeringto the animal in need thereof a compound of formula I or apharmaceutically acceptable salt thereof as described herein.

One embodiment provides a method of treating or preventing a bacterialinfection in an animal (e.g., a mammal such as a human) comprisingco-administering to the animal a compound of formula I or apharmaceutically acceptable salt thereof as described herein and one ormore antibacterial agents.

One embodiment provides a method of treating or preventing a bacterialinfection in an animal (e.g., a mammal such as a human) comprisingco-administering to the animal in need thereof a compound of formula Ior a pharmaceutically acceptable salt thereof as described herein andone or more antibacterial agents.

One embodiment provides a method of inhibiting a bacterial efflux pumpin an animal (e.g., a mammal such as a human) with a bacterial infectioncomprising administering to the animal a compound of formula I or apharmaceutically acceptable salt thereof as described herein.

One embodiment provides a method of treating or preventing a bacterialinfection in an animal (e.g., a mammal such as a human) infected withbacteria comprising co-administering to the animal a compound of formulaI or a pharmaceutically acceptable salt thereof as described herein andone or more antibacterial agents.

One embodiment provides a compound of formula I or a pharmaceuticallyacceptable salt thereof as described herein for use in medicaltreatment.

One embodiment provides a compound of formula I or a pharmaceuticallyacceptable salt thereof as described herein for the prophylactic ortherapeutic inhibition of a bacterial efflux pump for the treatment of abacterial infection.

One embodiment provides a compound of formula I or a pharmaceuticallyacceptable salt thereof as described herein which is used in combinationwith one or more antibacterial agents for the prophylactic ortherapeutic treatment of a bacterial infection.

One embodiment provides the use of a compound of formula I or apharmaceutically acceptable salt thereof as described herein for thepreparation of a medicament for inhibiting a bacterial efflux pump.

One embodiment provides the use of a compound of formula I or apharmaceutically acceptable salt thereof as described herein for thepreparation of a medicament for treating a bacterial infection in ananimal (e.g., a mammal such as a human).

One embodiment provides the use of a compound of formula I or apharmaceutically acceptable salt thereof as described herein for thepreparation of a medicament which is used in combination with one ormore antibacterial agents for treating a bacterial infection in ananimal (e.g., a mammal such as a human).

One embodiment provides processes and intermediates disclosed hereinthat are useful for preparing compounds of formula I or salts thereof.

DETAILED DESCRIPTION

The following definitions are used, unless otherwise described: halo orhalogen is fluoro, chloro, bromo, or iodo. Alkyl and alkoxy, etc. denoteboth straight and branched groups but reference to an individual radicalsuch as propyl embraces only the straight chain radical (a branchedchain isomer such as isopropyl being specifically referred to).

As used herein, the term “(C_(a)-C_(b))alkyl” wherein a and b areintegers refers to a straight or branched chain alkyl radical havingfrom a to b carbon atoms. Thus when a is 1 and b is 6, for example, theterm includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, t-butyl, n-pentyl and n-hexyl.

The term “aryl” as used herein refers to a single aromatic ring or amultiple condensed ring system wherein the ring atoms are carbon. Forexample, an aryl group can have 6 to 10 carbon atoms, or 6 to 12 carbonatoms. Aryl includes a phenyl radical. Aryl also includes multiplecondensed ring systems (e.g., ring systems comprising 2 rings) havingabout 9 to 12 carbon atoms or 9 to 10 carbon atoms in which at least onering is aromatic. Such multiple condensed ring systems may be optionallysubstituted with one or more (e.g., 1 or 2) oxo groups on any cycloalkylportion of the multiple condensed ring system. It is to be understoodthat the point of attachment of a multiple condensed ring system, asdefined above, can be at any position of the ring system including anaryl or a cycloalkyl portion of the ring. Typical aryl groups include,but are not limited to, phenyl, indenyl, naphthyl,1,2,3,4-tetrahydronaphthyl, anthracenyl, and the like.

The term “heteroaryl” as used herein refers to a single aromatic ring ora multiple condensed ring system. The term includes single aromaticrings of from about 1 to 6 carbon atoms and about 1-4 heteroatomsselected from the group consisting of oxygen, nitrogen and sulfur in therings. The sulfur and nitrogen atoms may also be present in an oxidizedform provided the ring is aromatic. Such rings include but are notlimited to pyridyl, pyrimidinyl, oxazolyl or furyl. The term alsoincludes multiple condensed ring systems (e.g. ring systems comprising 2rings) wherein a heteroaryl group, as defined above, can be condensedwith one or more heteroaryls (e.g., naphthyridinyl), heterocycles,(e.g., 1,2,3,4-tetrahydronaphthyridinyl), cycloalkyls (e.g.,5,6,7,8-tetrahydroquinolyl) or aryls (e.g. indazolyl) to form a multiplecondensed ring system. Such multiple condensed ring systems may beoptionally substituted with one or more (e.g., 1 or 2) oxo groups on thecycloalkyl or heterocycle portions of the condensed ring. In oneembodiment a monocyclic or bicyclic heteroaryl has 5 to 10 ring atomscomprising 1 to 9 carbon atoms and 1 to 4 heteroatoms. It is to beunderstood that the point of attachment of a multiple condensed ringsystem (as defined above for a heteroaryl) can be at any position of themultiple condensed ring system including a heteroaryl, heterocycle, arylor cycloalkyl portion of the multiple condensed ring system and at anysuitable atom of the multiple condensed ring system including a carbonatom and heteroatom (e.g., a nitrogen). Exemplary heteroaryls includebut are not limited to pyridyl, pyrrolyl, pyrazinyl, pyrimidinyl,pyridazinyl, pyrazolyl, thienyl, indolyl, imidazolyl, oxazolyl,thiazolyl, furyl, oxadiazolyl, thiadiazolyl, quinolyl, isoquinolyl,benzothiazolyl, benzoxazolyl, indazolyl, quinoxalyl, quinazolyl,5,6,7,8-tetrahydroisoquinolinyl, benzofuranyl, benzimidazolyl andthianaphthenyl.

The term “heterocyclyl” or “heterocycle” as used herein refers to asingle saturated or partially unsaturated ring or a multiple condensedring system. The term includes single saturated or partially unsaturatedrings (e.g., 3, 4, 5, 6 or 7-membered rings) from about 1 to 6 carbonatoms and from about 1 to 3 heteroatoms selected from the groupconsisting of oxygen, nitrogen and sulfur in the ring. The ring may besubstituted with one or more (e.g., 1, 2 or 3) oxo groups and the sulfurand nitrogen atoms may also be present in their oxidized forms. Suchrings include but are not limited to azetidinyl, tetrahydrofuranyl orpiperidinyl. It is to be understood that the point of attachment for aheterocycle can be at any suitable atom of the heterocycle Exemplaryheterocycles include, but are not limited to aziridinyl, azetidinyl,pyrrolidinyl, piperidinyl, homopiperidinyl, morpholinyl,thiomorpholinyl, piperazinyl, tetrahydrofuranyl, dihydrooxazolyl,tetrahydropyranyl and tetrahydrothiopyranyl.

The term “haloalkyl” includes an alkyl group as defined herein that issubstituted with one or more (e.g., 1, 2, 3, or 4) halo groups. Onespecific halo alkyl is a “(C₁-C₆)haloalkyl”.

The term cycloalkyl, carbocycle, or carbocyclyl includes saturated andpartially unsaturated carbocyclic ring systems. In one embodiment thecycloalkyl is a monocyclic carbocyclic ring. Such cycloalkyls include“(C₃-C₇)carbocyclyl” and “(C₃-C₈)cycloalkyl”.

Specific values listed below for radicals, substituents, and ranges, arefor illustration only; they do not exclude other defined values or othervalues within defined ranges for the radicals and substituents.

Specifically, (C₁-C₆)alkyl can be methyl, ethyl, propyl, isopropyl,butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl; (C₁-C₆)alkoxycan be methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy,sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy; (C₃-C₈)cycloalkyl can becyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; (C₁-C₆)haloalkylcan be iodomethyl, bromomethyl, chloromethyl, fluoromethyl,trifluoromethyl, 2-chloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, orpentafluoroethyl; aryl can be phenyl, indenyl, or naphthyl; andheteroaryl can be furyl, imidazolyl, triazolyl, triazinyl, oxazolyl,isoxazolyl, thiazolyl, isothiazoyl, pyrazolyl, pyrrolyl, pyrazinyl,tetrazolyl, pyridyl, (or its N-oxide), thienyl, pyrimidinyl (or itsN-oxide), indolyl, isoquinolyl (or its N-oxide) or quinolyl (or itsN-oxide).

It is understood that the embodiments provided below are for compoundsof formula I and all sub-formulas thereof (e.g., formulas Ia). It is tobe understood the two or more embodiments may be combined.

In one embodiment A is —C(═O)N(R^(a1))—R¹.

In one embodiment R^(a1) is hydrogen.

In one embodiment R² is hydrogen or (C₁-C₆)alkyl.

In one embodiment R² is hydrogen.

In one embodiment a compound of formula I is a compound of formula Ia:

or a salt thereof.

In one embodiment R³ is hydrogen, halo, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl,(C₁-C₄)alkoxy, or (C₁-C₄)haloalkoxy.

In one embodiment R³ is hydrogen.

In one embodiment R³ is hydrogen, halo, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl,(C₁-C₄)alkoxy, —OH, or (C₁-C₄)haloalkoxy.

In one embodiment R³ is hydrogen or —OH.

In one embodiment R³ is hydrogen, —OH, or methoxy.

In one embodiment R⁴ is hydrogen, aryl, or heteroaryl wherein the arylor heteroaryl is optionally substituted with one or more groupsindependently selected from the group consisting of halo, —OH, —NO₂,—CN, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, and(C₁-C₄)haloalkoxy.

In one embodiment R⁴ is phenyl wherein the phenyl is optionallysubstituted with one or more groups independently selected from thegroup consisting of halo, —OH, —NO₂, —CN, (C₁-C₄)alkyl,(C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, and (C₁-C₄)haloalkoxy.

In one embodiment R⁴ is phenyl wherein the phenyl is optionallysubstituted with one or more halo.

In one embodiment R⁴ is 4-fluorophenyl.

In one embodiment R⁴ is hydrogen or phenyl wherein the phenyl isoptionally substituted with one or more groups independently selectedfrom the group consisting of halo, —OH, —NO₂, —CN, (C₁-C₄)alkyl,(C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, and (C₁-C₄)haloalkoxy.

In one embodiment R⁴ is hydrogen or phenyl wherein the phenyl isoptionally substituted with one or more halo.

In one embodiment R⁴ is 4-fluorophenyl or hydrogen.

In one embodiment R⁵ is hydrogen, halo, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl,(C₁-C₄)alkoxy, or (C₁-C₄)haloalkoxy.

In one embodiment R⁵ is hydrogen.

In one embodiment R⁵ is hydrogen, halo, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl,(C₁-C₄)alkoxy, (C₁-C₄)haloalkoxy, or phenyl wherein the phenyl isoptionally substituted with one or more groups independently selectedfrom the group consisting of halo, —OH, —NO₂, —CN, (C₁-C₄)alkyl,(C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, and (C₁-C₄)haloalkoxy.

In one embodiment R⁵ is hydrogen or phenyl wherein the phenyl isoptionally substituted with one or more groups independently selectedfrom the group consisting of halo, —OH, —NO₂, —CN, (C₁-C₄)alkyl,(C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, and (C₁-C₄)haloalkoxy.

In one embodiment R⁵ is hydrogen or phenyl wherein the phenyl isoptionally substituted with one or more groups independently selectedfrom the group consisting of halo and (C₁-C₄)alkyl.

In one embodiment R⁵ is hydrogen, 4-fluorophenyl, phenyl, or3,5-dimethylphenyl.

In one embodiment R⁶ is hydrogen, halo, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl,(C₁-C₄)alkoxy, or (C₁-C₄)haloalkoxy.

In one embodiment R⁶ is hydrogen.

In one embodiment B is (C₃-C₇)carbocyclyl,(C₃-C₇)carbocyclyl-(C₁-C₄)alkyl-, aryl, aryl-(C₁-C₄)alkyl-, heteroaryl,or heteroaryl-(C₁-C₄)alkyl-, wherein any C₃-C₇)carbocyclyl,(C₃-C₇)carbocyclyl-(C₁-C₄)alkyl-, aryl, aryl-(C₁-C₄)alkyl-, heteroaryl,or heteroaryl-(C₁-C₄)alkyl- of B is optionally substituted with one ormore Z¹ groups.

In one embodiment B is (C₃-C₇)carbocyclyl, aryl, aryl-(C₁-C₄)alkyl-, orheteroaryl wherein any (C₃-C₇)carbocyclyl, aryl, aryl-(C₁-C₄)alkyl-, orheteroaryl, of B is optionally substituted with one or more Z¹ groups.

In one embodiment B is (C₃-C₇)carbocyclyl, phenyl, phenyl-(C₁-C₄)alkyl-,or 5-6 membered heteroaryl wherein any (C₃-C₇)carbocyclyl, phenyl,phenyl-(C₁-C₄)alkyl-, or 5-6 membered heteroaryl of B is optionallysubstituted with one or more Z¹ groups.

In one embodiment B is (C₃-C₇)carbocyclyl, phenyl, phenyl-(C₁-C₄)alkyl-,or 5-6 membered heteroaryl wherein any (C₃-C₇)carbocyclyl, phenyl,phenyl-(C₁-C₄)alkyl-, or 5-6 membered heteroaryl of B is optionallysubstituted with one or more Z¹ groups.

In one embodiment B is (C₃-C₇)carbocyclyl, phenyl, phenyl-(C₁-C₄)alkyl-,or 6 membered heteroaryl wherein any (C₃-C₇)carbocyclyl, phenyl,phenyl-(C₁-C₄)alkyl-, or 6 membered heteroaryl of B is optionallysubstituted with one or more Z¹ groups.

In one embodiment B is (C₃-C₇)carbocyclyl, phenyl, phenyl-(CH₂)—, orpyridinyl wherein any phenyl, phenyl-(CH₂)—, or pyridinyl of B isoptionally substituted with one or more Z¹ groups.

In one embodiment B is (C₃-C₇)carbocyclyl,(C₃-C₇)carbocyclyl-(C₁-C₄)alkyl-, (C₃-C₇)carbocyclyl-(C₂-C₆)alkynyl,aryl, aryl-(C₁-C₄)alkyl-, heteroaryl, or heteroaryl-(C₁-C₄)alkyl-,wherein any (C₃-C₇)carbocyclyl, (C₃-C₇)carbocyclyl-(C₁-C₄)alkyl-,(C₃-C₇)carbocyclyl-(C₂-C₆)alkynyl, aryl, aryl-(C₁-C₄)alkyl-, heteroaryl,or heteroaryl-(C₁-C₄)alkyl- of B is optionally substituted with one ormore Z groups.

In one embodiment B is (C₃-C₇)carbocyclyl,(C₃-C₇)carbocyclyl-(C₁-C₄)alkyl-, (C₃-C₇)carbocyclyl-(C₂-C₄)alkynyl,aryl, aryl-(C₁-C₄)alkyl-, or heteroaryl wherein any (C₃-C₇)carbocyclyl,aryl, aryl-(C₁-C₄)alkyl-, or heteroaryl, of B is optionally substitutedwith one or more Z groups.

In one embodiment B is (C₃-C₇)carbocyclyl,(C₃-C₇)carbocyclyl-(C₁-C₄)alkyl-, (C₃-C₇)carbocyclyl-(C₂-C₆)alkynyl,phenyl, phenyl-(C₁-C₄)alkyl-, or 5-6 membered heteroaryl wherein any(C₃-C₇)carbocyclyl, (C₃-C₇)carbocyclyl-(C₁-C₄)alkyl-,(C₃-C₇)carbocyclyl-(C₂-C₆)alkynyl, phenyl, phenyl-(C₁-C₄)alkyl-, or 5-6membered heteroaryl of B is optionally substituted with one or more Z¹groups.

In one embodiment B is (C₃-C₇)carbocyclyl, phenyl, phenyl-(C₁-C₄)alkyl-,or 5-6 membered heteroaryl wherein any (C₃-C₇)carbocyclyl, phenyl,phenyl-(C₁-C₄)alkyl-, or 5-6 membered heteroaryl of B is optionallysubstituted with one or more Z¹ groups.

In one embodiment B is (C₃-C₇)carbocyclyl, phenyl, phenyl-(C₁-C₄)alkyl-,(C₃-C₇)carbocyclylethynyl, (C₃-C₇)carbocyclylethyl, or 6 memberedheteroaryl wherein any (C₃-C₇)carbocyclyl, phenyl, phenyl-(C₁-C₄)alkyl-,C₃-C₇)carbocyclylethynyl, (C₃-C₇)carbocyclylethyl, or 6 memberedheteroaryl of B is optionally substituted with one or more Z¹ groups.

In one embodiment B is (C₃-C₇)carbocyclyl, phenyl, phenyl-(CH₂)—,(C₃-C₇)carbocyclylethynyl, (C₃-C₇)carbocyclylethyl or pyridinyl whereinany phenyl, phenyl-(CH₂)—, C₃-C₇)carbocyclylethynyl,(C₃-C₇)carbocyclylethyl or pyridinyl of B is optionally substituted withone or more Z¹ groups.

In one embodiment B is 4-fluorophenyl, cyclopropyl, benzyl, pyrdin-4-yl,4-hydroxyphenyl, 4-trifluoromethylphenyl, 4-benzoic acid,3-benzyloxyphenyl, 3-hydroxyphenyl, phenyl, 3,5-dimethylphenyl,2-cylopropylethynyl, 2- or cyclpropylethyl.

In one embodiment each Z¹ is independently halo, —OH, or(C₁-C₄)haloalkyl.

In one embodiment each Z¹ is independently halo, —OH, —NO₂, —CN,(C₁-C₄)alkyl, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, or (C₁-C₄)haloalkoxy;

In one embodiment each Z¹ is independently halo, —OH, —CO₂H, benzyloxy,or (C₁-C₄)haloalkyl.

In one embodiment B is 4-fluorophenyl, cyclopropyl, benzyl, pyrdin-4-yl,4-hydroxyphenyl, or 4-trifluoromethylphenyl.

In one embodiment R¹ is (C₁-C₁₄)alkyl substituted with one or moregroups independently selected from —NR^(b2)R^(c2) and wherein the(C₁-C₁₄)alkyl is optionally substituted with one or more(C₃-C₇)carbocyclyl.

In one embodiment R¹ is (C₂-C₁₀)alkyl substituted with one or moregroups independently selected from —NR^(b2)R^(c2) and wherein the(C₂-C₁₀)alkyl is optionally substituted with one or more(C₃-C₇)carbocyclyl.

In one embodiment R¹ is (C₄-C₈)alkyl substituted with two or more groupsindependently selected from —NR^(b2)R^(c2).

In one embodiment R^(b2) and R² are each hydrogen.

In one embodiment R¹ is a 4-7 membered monocyclicheterocyclyl-(C₁-C₄)alkyl-, wherein the 4-7 membered monocyclicheterocyclyl-(C₁-C₄)alkyl- is substituted with one or more groupsindependently selected from the group consisting of Z and —(C₁-C₆)alkylsubstituted with one or more Z, wherein each Z is independently selectedfrom the group consisting of —NR^(b3)R^(c3), —NHNH₂,—C(═NR^(a3))(NR^(b3)R^(c3)), —NR^(a3)C(═NR^(a3))(R^(d3)), andNR^(a3)C(═NR^(a3))(NR^(b3)R^(c3)) and wherein the 4-7 memberedmonocyclic heterocyclyl-(C₁-C₄)alkyl- is optionally substituted with oneor more (C₁-C₆)alkyl.

In one embodiment R¹ is a 4-7 membered monocyclicheterocyclyl-(C₁-C₄)alkyl-, wherein the 4-7 membered monocyclicheterocyclyl-(C₁-C₄)alkyl- is substituted with one or more groupsindependently selected from the group consisting of Z and (C₁-C₆)alkylsubstituted with one or more Z, wherein each Z is independently—NR^(b3)R^(c3) and wherein the 4-7 membered monocyclicheterocyclyl-(C₁-C₄)alkyl- is optionally substituted with one or more(C₁-C₆)alkyl.

In one embodiment R¹ is pyrrolidinyl-(C₁-C₄)alkyl-, wherein thepyrrolidinyl-(C₁-C₄)alkyl- is substituted with one or more groupsindependently selected from the group consisting of Z and —(C₁-C₆)alkylsubstituted with one or more Z, wherein each Z is independently—NR^(b3)R^(c3) and wherein is pyrrolidinyl-(C₁-C₄)alkyl- is optionallysubstituted independently with one or more (C₁-C₆)alkyl In oneembodiment R¹ is pyrrolidinyl-(CH₂)—, wherein the pyrrolidinyl-(CH₂)— issubstituted with one or more groups independently selected from thegroup consisting of Z and —(C₁-C₆)alkyl substituted with one or more Z,wherein each Z is independently —NR^(b3)R^(c3) and wherein thepyrrolidinyl-(CH₂)— is optionally substituted independently with one ormore (C₁-C₆)alkyl.

In one embodiment R¹ is pyrrolidinyl-(CH₂)—, wherein thepyrrolidinyl-(CH₂)— is substituted on the pyrrolidinyl with—(C₁-C₆)alkyl substituted with one or more —NR^(b3)R^(c3) In oneembodiment R^(b3) and R³ are each hydrogen. In one embodiment R¹ is:

In one embodiment A is:

One embodiment provides a compound that is:

or a salt thereof (or a pharmaceutically acceptable salt thereof).

One embodiment provides a compound that is:

One embodiment provides a method for identifying a test compound capableof inhibiting a bacterial efflux inhibitor, comprising

-   -   1) contacting bacteria with a sub-inhibitory concentration of an        antibiotic;    -   2) contacting the bacteria, either sequentially or        simultaneously, with a) an inhibitory concentration of the        antibiotic; and b) a test compound; and    -   3) quantifying the minimum inhibitory concentration (MIC) of the        antibiotic, wherein a MIC that is lower than the intrinsic MIC        of the antibiotic alone indicates the test compound is effective        to inhibit a bacterial efflux pump inhibitor.

In certain embodiments, steps 1 and 2 are separated by about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 38, 39, 40, 41,42, 43, 44, 45, 46, 47 or 48 hours. In certain embodiments, steps 1 and2 are separated by about 12 hours. In certain embodiments, steps 1 and 2are separated by about 24 hours.

One embodiment provides a method for identifying a test compound capableof inhibiting a bacterial efflux inhibitor, comprising

-   -   1) contacting bacteria with a sub-inhibitory concentration of an        antibiotic;    -   2) contacting a first subset of the bacteria with an inhibitory        concentration of the antibiotic;    -   3) contacting a second subset of the bacteria, either        sequentially or simultaneously, with a) an inhibitory        concentration of the antibiotic; and b) a test compound; and    -   4) quantifying the minimum inhibitory concentration (MIC) of the        antibiotic for the first subset of bacteria and the second        subset of bacteria, wherein a lower MIC in the second subset        indicates the test compound is effective to inhibit a bacterial        efflux pump inhibitor.

In certain embodiments, steps 1 and 2 and/or 1 and 3 are separated byabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 3738, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 hours. In certainembodiments, steps 1 and 2 are separated by about 12 hours. In certainembodiments, steps 1 and 2 are separated by about 24 hours. In certainembodiments, steps 1 and 3 are separated by about 12 hours. In certainembodiments, steps 1 and 3 are separated by about 24 hours. In certainembodiments, steps 2 and 3 are performed at substantially the same time(e.g., at about less than 10, 30, 60, 90 or 120 seconds apart, or about3, 4 or 5 minutes apart).

One embodiment provides a method for identifying a test compound capableof lowering the minimum inhibitory concentration (MIC) of an antibiotic,comprising

-   -   1) contacting bacteria with a sub-inhibitory concentration of        the antibiotic;    -   2) contacting the bacteria, either sequentially or        simultaneously, with a) an inhibitory concentration of the        antibiotic; and b) the test compound; and    -   3) quantifying the minimum inhibitory concentration (MIC) of the        antibiotic, wherein a MIC that is lower than the intrinsic MIC        of the antibiotic indicates the test compound is effective to        lower the MIC of the antibiotic.

In certain embodiments, steps 1 and 2 are separated by about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 38, 39, 40, 41,42, 43, 44, 45, 46, 47 or 48 hours. In certain embodiments, steps 1 and2 are separated by about 12 hours. In certain embodiments, steps 1 and 2are separated by about 24 hours.

One embodiment provides a method for identifying a test compound capableof lowering the minimum inhibitory concentration (MIC) of an antibiotic,comprising

-   -   1) contacting bacteria with a sub-inhibitory concentration of an        antibiotic;    -   2) contacting a first subset of the bacteria with an inhibitory        concentration of the antibiotic;    -   3) contacting a second subset of the bacteria, either        sequentially or simultaneously, with a) an inhibitory        concentration of the antibiotic; and b) a test compound; and    -   4) quantifying the minimum inhibitory concentration (MIC) of the        antibiotic for the first subset of bacteria and the second        subset of bacteria, wherein a lower MIC in the second subset        indicates the test compound is effective to lower the MIC of the        antibiotic.

In certain embodiments, steps 1 and 2 and/or 1 and 3 are separated byabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 3738, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 hours. In certainembodiments, steps 1 and 2 are separated by about 12 hours. In certainembodiments, steps 1 and 2 are separated by about 24 hours. In certainembodiments, steps 1 and 3 are separated by about 12 hours. In certainembodiments, steps 1 and 3 are separated by about 24 hours. In certainembodiments, steps 2 and 3 are performed at substantially the same time(e.g., at about less than 10, 30, 60, 90 or 120 seconds apart, or about3, 4 or 5 minutes apart).

Such methods may also be used to determine synergy between a testcompound and an antibiotic.

As used herein, the term “minimum inhibitory concentration (MIC)” refersto the lowest concentration of a compound (e.g., an antibiotic) thatprevents visible growth of a bacterium. Assays for measuring the MIC ofa compound are known in the art, for example, as described herein. Asused herein, the term “intrinsic MIC” refers the MIC of a compound(e.g., an antibiotic) for the particular bacterial species that has notbeen pre-exposed to the compound.

As used herein, the term “sub-inhibitory concentration” refers to aconcentration of the antibiotic that does not reduce the visible growthof the bacteria. In certain embodiments, the sub-inhibitoryconcentration is ½×MIC of the antibiotic. In certain embodiments, thesub-inhibitory concentration of the antibiotic is a concentration thatis capable of inducing the expression of one or more efflux pumps in thebacteria.

As used herein, the term “inhibitory concentration” refers to aconcentration of the antibiotic that reduces the visible growth of thebacteria. In certain embodiments, this concentration is the intrinsicMIC of the antibiotic.

In certain embodiments, the bacteria are a species of bacteria describedherein. In certain embodiments, the bacteria are P. aeruginosa.

In certain embodiments, the antibiotic is an antibiotic describedherein. In certain embodiments, the antibiotic is cefepime,clarithromycin, or levofloxacin.

In certain embodiments, the test compound is a compound describedherein, such as a compound of formula I, an efflux pump inhibitor (EPI),etc.

One embodiment provides a method of identifying a combination of a testcompound and an antibiotic that is capable of treating septicemia in ananimal comprising:

-   -   1) administering the test compound to the animal intravenously;    -   2) administering the antibiotic to the animal either orally or        intravenously;    -   3) administering the test compound to the animal subcutaneously;    -   4) administering the antibiotic to the animal either orally or        intravenously; and    -   5) evaluating the animal for symptoms of septicemia, wherein a        reduction in symptoms indicates the combination is effective to        treat septicemia.

In certain embodiments, each administration is independently separatedby approximately about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40,45, 50, 55 or 60 min. In certain embodiments, each administration isseparated by about 5 minutes.

In certain embodiments, the method further comprises repeating steps1-4. For example, in certain embodiments, steps 1-4 are repeated 24hours after the antibiotic has been administered for the second time.

In certain embodiments, the combination of the test compound andantibiotic is a synergistic combination.

In certain embodiments, the animal is a non-human animal. For example,in certain embodiments, the animal is a mouse.

In certain embodiments, the antibiotic is an antibiotic describedherein. In certain embodiments, the antibiotic is cefepime,clarithromycin, or levofloxacin.

In certain embodiments, the test compound is a compound describedherein, such as a compound of formula I, an efflux pump inhibitor (EPI),etc.

One embodiment provides a method described herein for identifying acompound capable of inhibiting a bacterial efflux pump inhibitor (e.g.,using an assay described in the Examples).

One embodiment provides a compound of formula I:

wherein:

A is —C(═O)N(R^(a1))—R¹, —(C₁-C₃)alkyl-C(═O)N(R^(a1))R¹,—(C₁-C₃)alkyl-O—R¹, —O—R¹, —(C₁-C₃)alkyl-N(R^(a1))—R¹, —N(R^(a1))—R¹, orR¹;

B is (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy,(C₁-C₄)haloalkoxy, (C₃-C₇)carbocyclyl, (C₃-C₇)carbocyclyl-(C₁-C₄)alkyl-,aryl, aryl-(C₁-C₄)alkyl-, heteroaryl, heteroaryl-(C₁-C₄)alkyl-, 3-7membered-monocyclic-heterocycle, or 3-7membered-monocyclic-heterocycle-(C₁-C₄)alkyl- wherein any(C₃-C₇)carbocyclyl, (C₃-C₇)carbocyclyl-(C₁-C₄)alkyl-, aryl,aryl-(C₁-C₄)alkyl-, heteroaryl, heteroaryl-(C₁-C₄)alkyl-, 3-7membered-monocyclic-heterocycle, or 3-7membered-monocyclic-heterocycle-(C₁-C₄)alkyl- of B is optionallysubstituted with one or more Z¹ groups;

each R¹ is independently:

(a) (C₁-C₁₄)alkyl substituted with one or more groups selected from thegroup consisting of —NR^(b2)R^(c2), —NHNH₂, —C(═NR^(a2))(NR^(b2)R^(c2)),—NR^(a2)C(═NR^(a2))(R^(d2)), and —NR^(a2)C(═NR^(a2))(NR^(b2)R^(c2)) andwherein (C₁-C₁₄)alkyl is optionally substituted independently with oneor more (C₁-C₄)alkyl or (C₃-C₇)carbocyclyl; or

(b) (C₃-C₇)carbocyclyl, (C₃-C₇)carbocyclyl-(C₁-C₄)alkyl-, 4-7 memberedmonocyclic heterocyclyl, or 4-7 membered monocyclicheterocyclyl-(C₁-C₄)alkyl-, wherein each (C₃-C₇)carbocyclyl or(C₃-C₇)carbocyclyl-(C₁-C₄)alkyl- is independently substituted with oneor more groups selected from the group consisting of Z and —(C₁-C₆)alkylsubstituted with one or more Z, and wherein each 4-7 membered monocyclicheterocyclyl or 4-7 membered monocyclic heterocyclyl-(C₁-C₄)alkyl- isindependently optionally substituted with one or more groups selectedfrom the group consisting of Z and —(C₁-C₆)alkyl substituted with one ormore Z, wherein each Z is independently selected from the groupconsisting of —NR^(b3)R^(c3), —NHNH₂, —C(═NR^(a3))(NR^(b3)R^(c3)),—NR^(a3)C(═NR^(a3))(R^(d3)), and —NR^(a3)C(═NR^(a3))(NR^(b3)R^(c3)) andwherein each (C₃-C₇)carbocyclyl, (C₃-C₇)carbocyclyl-(C₁-C₄)alkyl-, 4-7membered monocyclic heterocyclyl, or 4-7 membered monocyclicheterocyclyl-(C₁-C₄)alkyl-, is optionally substituted independently withone or more (C₁-C₄)alkyl;

R² is hydrogen, (C₁-C₄)alkyl or phenyl(C₁-C₃)alkyl-, wherein the phenylis optionally substituted with one or more (C₁-C₄)alkyl, —O(C₁-C₄)alkyl,halogen, or —NO₂;

R³ is hydrogen, halo, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy,(C₁-C₄)haloalkoxy, aryl, or heteroaryl wherein the aryl or heteroaryl isoptionally substituted with one or more groups independently selectedfrom the group consisting of halo, —OH, —NO₂, —CN, (C₁-C₄)alkyl,(C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, and (C₁-C₄)haloalkoxy;

R⁴ is hydrogen, halo, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy,(C₁-C₄)haloalkoxy, aryl, or heteroaryl wherein the aryl or heteroaryl isoptionally substituted with one or more groups independently selectedfrom the group consisting of halo, —OH, —NO₂, —CN, (C₁-C₄)alkyl,(C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, and (C₁-C₄)haloalkoxy;

R⁵ is hydrogen, halo, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy,(C₁-C₄)haloalkoxy, aryl, or heteroaryl wherein the aryl or heteroaryl isoptionally substituted with one or more groups independently selectedfrom the group consisting of halo, —OH, —NO₂, —CN, (C₁-C₄)alkyl,(C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, and (C₁-C₄)haloalkoxy;

R⁶ is hydrogen, halo, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy,(C₁-C₄)haloalkoxy, aryl, or heteroaryl wherein the aryl or heteroaryl isoptionally substituted with one or more groups independently selectedfrom the group consisting of halo, —OH, —NO₂, —CN, (C₁-C₄)alkyl,(C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, and (C₁-C₄)haloalkoxy;

each Z¹ is independently halo, —OH, —NO₂, —CN, (C₁-C₄)alkyl,(C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, or (C₁-C₄)haloalkoxy;

each R^(a1) is independently hydrogen, (C₁-C₄)alkyl or(C₃-C₇)carbocyclyl;

each R^(a2) is independently hydrogen, (C₁-C₄)alkyl or(C₃-C₇)carbocyclyl;

each R^(b2) and R^(c2) is independently hydrogen, (C₁-C₄)alkyl or(C₃-C₇)carbocyclyl;

R^(d2) is (C₁-C₄)alkyl or (C₃-C₇)carbocyclyl;

each R^(a3) is independently hydrogen (C₁-C₄)alkyl or(C₃-C₇)carbocyclyl;

each R^(b3) and R³ is independently hydrogen (C₁-C₄)alkyl or(C₃-C₇)carbocyclyl; and

R^(d3) is (C₁-C₄)alkyl or (C₃-C₇)carbocyclyl;

or a salt thereof.

One embodiment provides a compound of formula I:

wherein:

A is —C(═O)N(R^(a1))—R¹, —(C₁-C₃)alkyl-C(═O)N(R^(a1))R¹,—(C₁-C₃)alkyl-O—R¹, —O—R¹, —(C₁-C₃)alkyl-N(R^(a1))—R¹, —N(R^(a1))—R¹, orR¹;

B is (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy,(C₁-C₄)haloalkoxy, (C₃-C₇)carbocyclyl, (C₃-C₇)carbocyclyl-(C₁-C₄)alkyl-,aryl, aryl-(C₁-C₄)alkyl-, heteroaryl, heteroaryl-(C₁-C₄)alkyl-, 3-7membered-monocyclic-heterocycle, or 3-7membered-monocyclic-heterocycle-(C₁-C₄)alkyl- wherein any(C₃-C₇)carbocyclyl, (C₃-C₇)carbocyclyl-(C₁-C₄)alkyl-, aryl,aryl-(C₁-C₄)alkyl-, heteroaryl, heteroaryl-(C₁-C₄)alkyl-, 3-7membered-monocyclic-heterocycle, or 3-7membered-monocyclic-heterocycle-(C₁-C₄)alkyl- of B is optionallysubstituted with one or more Z¹ groups;

each R¹ is independently:

(a) (C₁-C₁₄)alkyl substituted with one or more groups selected from thegroup consisting of —NR^(b2)R^(c2), —NHNH₂, —C(═NR^(a2))(NR^(b2)R^(c2)),—NR^(a2)C(═NR^(a2))(R^(d2)), and —NR^(a2)C(═NR^(a2))(NR^(b2)R^(c2)) andwherein (C₁-C₁₄)alkyl is optionally substituted independently with oneor more halo, (C₁-C₄)alkyl or (C₃-C₇)carbocyclyl; or

(b) (C₃-C₇)carbocyclyl, (C₃-C₇)carbocyclyl-(C₁-C₄)alkyl-, 4-7 memberedmonocyclic heterocyclyl, or 4-7 membered monocyclicheterocyclyl-(C₁-C₄)alkyl-, wherein each (C₃-C₇)carbocyclyl or(C₃-C₇)carbocyclyl-(C₁-C₄)alkyl- is independently substituted with oneor more Z² or Z³, and wherein each 4-7 membered monocyclic heterocyclylor 4-7 membered monocyclic heterocyclyl-(C₁-C₄)alkyl- is independentlyoptionally substituted with one or more Z² or Z³, and wherein any(C₃-C₇)carbocyclyl, (C₃-C₇)carbocyclyl-(C₁-C₄)alkyl-, 4-7 memberedmonocyclic heterocyclyl, or 4-7 membered monocyclicheterocyclyl-(C₁-C₄)alkyl- of R¹ is optionally substituted independentlywith one or more halo, (C₁-C₄)alkyl or (C₃-C₇)carbocyclyl;

R² is hydrogen, (C₁-C₄)alkyl or phenyl(C₁-C₃)alkyl-, wherein the phenylis optionally substituted with one or more (C₁-C₄)alkyl, —O(C₁-C₄)alkyl,halogen, or —NO₂;

R³ is hydrogen, halo, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy,(C₁-C₄)haloalkoxy, aryl, or heteroaryl wherein the aryl or heteroaryl isoptionally substituted with one or more groups independently selectedfrom the group consisting of halo, —OH, —NO₂, —CN, (C₁-C₄)alkyl,(C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, and (C₁-C₄)haloalkoxy;

R⁴ is hydrogen, halo, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy,(C₁-C₄)haloalkoxy, aryl, or heteroaryl wherein the aryl or heteroaryl isoptionally substituted with one or more groups independently selectedfrom the group consisting of halo, —OH, —NO₂, —CN, (C₁-C₄)alkyl,(C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, and (C₁-C₄)haloalkoxy;

R⁵ is hydrogen, halo, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy,(C₁-C₄)haloalkoxy, aryl, or heteroaryl wherein the aryl or heteroaryl isoptionally substituted with one or more groups independently selectedfrom the group consisting of halo, —OH, —NO₂, —CN, (C₁-C₄)alkyl,(C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, and (C₁-C₄)haloalkoxy;

R⁶ is hydrogen, halo, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy,(C₁-C₄)haloalkoxy, aryl, or heteroaryl wherein the aryl or heteroaryl isoptionally substituted with one or more groups independently selectedfrom the group consisting of halo, —OH, —NO₂, —CN, (C₁-C₄)alkyl,(C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, and (C₁-C₄)haloalkoxy;

each Z¹ is independently halo, —OH, —NO₂, —CN, (C₁-C₄)alkyl,(C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, or (C₁-C₄)haloalkoxy;

each Z² is independently selected from the group consisting of—NR^(b3)R^(c3), —NHNH₂, —C(═NR^(a3))(NR^(b3)R^(c3)),—NR^(a3)C(═NR^(a3))(R^(d3)), and —NR^(a3)C(═NR^(a3))(NR^(b3)R^(c3))

each Z³ is independently —(C₁-C₆)alkyl substituted with one or more Z²and optionally optionally substituted with one or more Z⁴;

each Z⁴ is independently halo or (C₃-C₇)carbocyclyl;

each R^(a1) is independently hydrogen, (C₁-C₄)alkyl or(C₃-C₇)carbocyclyl;

each R^(a2) is independently hydrogen, (C₁-C₄)alkyl or(C₃-C₇)carbocyclyl;

each R^(b2) and R² is independently hydrogen, (C₁-C₄)alkyl or(C₃-C₇)carbocyclyl;

R^(d2) is (C₁-C₄)alkyl or (C₃-C₇)carbocyclyl;

each R^(a3) is independently hydrogen (C₁-C₄)alkyl or(C₃-C₇)carbocyclyl;

each R^(b3) and R³ is independently hydrogen (C₁-C₄)alkyl or(C₃-C₇)carbocyclyl; and

R^(d3) is (C₁-C₄)alkyl or (C₃-C₇)carbocyclyl;

or a salt thereof.

Generally, compounds of formula I as well as synthetic intermediatesthat can be used for preparing compounds of formula I can be prepared asillustrated in the following General Methods and Schemes. It isunderstood that variable groups shown below (e.g., R¹, R², R³, R⁴, R⁵,R⁶) can represent the final corresponding groups present in a compoundof formula I or that these groups can represent groups that can beconverted to the final corresponding groups present in a compound offormula I at a convenient point in a synthetic sequence. For example,the variable groups can contain one or more protecting groups that canbe removed at a convenient point in a synthetic sequence to provide thefinal corresponding groups in the compound of formula I.

Schemes 1 and 2 illustrate some general methods for the preparation ofsubstituted indole carboxamides.

The compounds disclosed herein are bacterial efflux pump inhibitors. Anefflux pump inhibitor is a compound that interferes with the ability ofan efflux pump to export a substrate. The inhibitor may have intrinsicantibacterial properties of its own. The compounds disclosed herein maybe useful for treating bacterial infections (e.g., gram negative andgram positive) when administered with an antibacterial agent.

In one embodiment the bacterial infection being treated is aGram-negative bacterial strain infection. In one embodiment theGram-negative bacterial strain is selected from the group consisting ofAcinetobacter baumannii, Acinetobacter calcoaceticus, Acinetobacterhaemolyticus, Acinetobacter lwoffi, Actinobacillusactinomycetemcomitans, Aeromonas hydrophilia, Aggregatibacteractinomycetemcomitans, Agrobacterium tumefaciens, Bacteroidesdistasonis, Bacteroides eggerthii, Bacteroides forsythus, Bacteroidesfragilis, Bacteroides ovalus, Bacteroides splanchnicus, Bacteroidesthetaiotaomicron, Bacteroides uniformis, Bacteroides vulgatus,Bordetella bronchiseptica, Bordetella parapertussis, Bordetellapertussis, Borrelia burgdorferi, Branhamella catarrhalis, Burkholderiacepacia, Campylobacter coli, Campylobacter fetus, Campylobacterjejuni,Caulobacter crescentus, Chlamydia trachomatis, Citrobacter diversus,Citrobacter freundii, Enterobacter aerogenes, Enterobacter asburiae,Enterobacter cloacae, Enterobacter sakazakii, Escherchia coli,Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis,Haemophilus ducreyi, Haemophilus haemolyticus, Haemophilus influenzae,Haemophilus parahaemolyticus, Haemophilus parainfluenzae, Helicobacterpylori, Kingella denitrificans, Kingella indologenes, Kingella kingae,Kingella oralis, Klebsiella oxytoca, Klebsiella pneumoniae, Klebsiellarhinoscleromatis, Legionella pneumophila, Listeria monocytogenes,Moraxella bovis, Moraxella catarrhalis, Moraxella lacunata, Morganellamorganii, Neisseria gonorrhoeae, Neisseria meningitidis, Pantoeaagglomerans, Pasteurella canis, Pasteurella haemolytica, Pasteurellamultocida, Pasteurella tularensis, Porphyromonas gingivalis, Proteusmirabilis, Proteus vulgaris, Providencia alcalifaciens, Providenciarettgeri, Providencia stuartii, Pseudomonas acidovorans, Pseudomonasaeruginosa, Pseudomonas alcaligenes, Pseudomonas fluorescens,Pseudomonas putida, Salmonella enteriditis, Salmonella paratyphi,Salmonella typhi, Salmonella typhimurium, Serratia marcescens, Shigelladysenteriae, Shigella jlexneri, Shigella sonnei, Stenotrophomonasmaltophilla, Veillonella parvula, Vibrio cholerae, Vibrioparahaemolyticus, Yersinia enterocolitica, Yersinia intermedia, Yersiniapestis and Yersinia pseudotuberculosis.

In one embodiment the bacterial infection being treated is aGram-positive bacterial strain infection. In one embodiment theGram-positive bacterial strain is selected from the group consisting ofActinomyces naeslundii, Actinomyces viscosus, Bacillus anthracis,Bacillus cereus, Bacillus subtilis, Clostridium difficile,Corynebacterium diphtheriae, Corynebacterium ulcerans, Enterococcusfaecalis, Enterococcus faecium, Micrococcus luteus, Mycobacterium avium,Mycobacterium intracellulare, Mycobacterium leprae, Mycobacteriumtuberculosis, Propionibacterium acnes, Staphylococcus aureus,Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcushominis, Staphylococcus hyicus, Staphylococcus intermedius,Staphylococcus saccharolyticus, Staphylococcus saprophyticus,Streptococcus agalactiae, Streptococcus mutans, Streptococcuspneumoniae, Streptococcus pyogenes, Streptococcus salivarius andStreptococcus sanguis.

The compositions can, if desired, also contain other active therapeuticagents, such as a narcotic, a non-steroid anti-inflammatory drug(NSAID), an analgesic, an anesthetic, a sedative, a local anesthetic, aneuromuscular blocker, an anti-cancer, an antimicrobial (for example, anaminoglycoside, an antifungal, an antiparasitic, an antiviral, acarbapenem, a cephalosporin (e.g., cefepime), a fluoroquinolone, amacrolide, a penicillin, a sulfonamide, a tetracycline, anotherantimicrobial), an anti-psoriatic, a corticosteriod, an anabolicsteroid, a diabetes-related agent, a mineral, a nutritional, a thyroidagent, a vitamin, a calcium-related hormone, an antidiarrheal, ananti-tussive, an anti-emetic, an anti-ulcer, a laxative, ananticoagulant, an erythropoietin (for example, epoetin alpha), afilgrastim (for example, G-CSF, Neupogen), a sargramostim (GM-CSF,Leukine), an immunization, an immunoglobulin, an immunosuppressive (forexample, basiliximab, cyclosporine, daclizumab), a growth hormone, ahormone replacement drug, an estrogen receptor modulator, a mydriatic, acycloplegic, an alkylating agent, an anti-metabolite, a mitoticinhibitor, a radiopharmaceutical, an anti-depressant, an anti-manicagent, an anti-psychotic, an anxiolytic, a hypnotic, a sympathomimetic,a stimulant, donepezil, tacrine, an asthma medication, a beta agonist,an inhaled steroid, a leukotriene inhibitor, a methylxanthine, acromolyn, an epinephrine or analog thereof, dornase alpha (Pulmozyme), acytokine, or any combination thereof.

In one embodiment the antibacterial agent is selected from quinolones,tetracyclines, glycopeptides, aminoglycosides, β-lactams, rifamycins,macrolides, ketolides, oxazolidinones, coumermycins, andchloramphenicol.

It will be appreciated that compounds of the invention having a chiralcenter may exist in and be isolated in optically active and racemicforms. Some compounds may exhibit polymorphism. It is to be understoodthat the present invention encompasses any racemic, optically-active,polymorphic, or stereoisomeric form, or mixtures thereof, of a compoundof the invention, which possess the useful properties described herein,it being well known in the art how to prepare optically active forms(for example, by resolution of the racemic form by recrystallizationtechniques, by synthesis from optically-active starting materials, bychiral synthesis, or by chromatographic separation using a chiralstationary phase. When a bond in a compound formula herein is drawn in anon-stereochemical manner (e.g. flat), the atom to which the bond isattached includes all stereochemical possibilities.

When a bond in a compound formula herein is drawn in a definedstereochemical manner (e.g. bold, bold-wedge, dashed or dashed-wedge),it is to be understood that the atom to which the stereochemical bond isattached is enriched in the absolute stereoisomer depicted unlessotherwise noted. In one embodiment, the compound may be at least 51% theabsolute stereoisomer depicted. In another embodiment, the compound maybe at least 60% the absolute stereoisomer depicted. In anotherembodiment, the compound may be at least 80% the absolute stereoisomerdepicted. In another embodiment, the compound may be at least 90% theabsolute stereoisomer depicted. In another embodiment, the compound maybe at least 95 the absolute stereoisomer depicted. In anotherembodiment, the compound may be at least 99% the absolute stereoisomerdepicted.

It will also be appreciated by those skilled in the art that certaincompounds of the invention can exist in more than one tautomeric form.For example, a substituent of formula —NH—C(═O)H in a compound offormula (I) could exist in tautomeric form as —N═C(OH)H. The presentinvention encompasses all tautomeric forms of a compound of formula I aswell as mixtures thereof that can exist in equilibrium with non-chargedand charged entities depending upon pH, which possess the usefulproperties described herein

In cases where compounds are sufficiently basic or acidic, a salt of acompound of formula I can be useful as an intermediate for isolating orpurifying a compound of formula I. Additionally, administration of acompound of formula I as a pharmaceutically acceptable acid or base saltmay be appropriate. Examples of pharmaceutically acceptable salts areorganic acid addition salts formed with acids which form a physiologicalacceptable anion, for example, tosylate, methanesulfonate, acetate,citrate, malonate, tartrate, succinate, fumarate, benzoate, ascorbate,α-ketoglutarate, and α-glycerophosphate. Suitable inorganic salts mayalso be formed, including hydrochloride, sulfate, nitrate, bicarbonate,and carbonate salts. Salts may be obtained using standard procedureswell known in the art, for example by reacting a sufficiently basiccompound such as an amine with a suitable acid affording thecorresponding anion. Alkali metal (for example, sodium, potassium orlithium) or alkaline earth metal (for example calcium) salts ofcarboxylic acids can also be made.

Pharmaceutically suitable counterions include pharmaceutically suitablecations and pharmaceutically suitable anions that are well known in theart. Examples of pharmaceutically suitable anions include, but are notlimited to those described above (e.g. physiologically acceptableanions) including Cl⁻, Br⁻, I⁻, CH₃SO₃ ⁻, H₂PO₄ ⁻, CF₃SO₃ ⁻,p-CH₃C₆H₄SO₃ ⁻, citrate, tartrate, phosphate, malate, fumarate, formate,or acetate.

It will be appreciated by those skilled in the art that a compound ofthe invention comprising a counterion can be converted to a compound ofthe invention comprising a different counterion. Such a conversion canbe accomplished using a variety of well-known techniques and materialsincluding but not limited to ion exchange resins, ion exchangechromatography and selective crystallization.

The compounds of formula I can be formulated as pharmaceuticalcompositions and administered to a mammalian host, such as a humanpatient in a variety of forms adapted to the chosen route ofadministration, i.e., orally or parenterally, by intravenous,intramuscular, topical or subcutaneous routes. For oral administrationthe compounds can be formulated as a solid dosage form with or withoutan enteric coating.

Thus, the present compounds may be systemically administered, e.g.,orally, in combination with a pharmaceutically acceptable vehicle suchas an inert diluent, excipient or an assimilable edible carrier. Theymay be enclosed in hard or soft shell gelatin capsules, may becompressed into tablets, or may be incorporated directly with the foodof the patient's diet. For oral therapeutic administration, the activecompound may be combined with one or more excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations should contain at least 0.1% of active compound. Thepercentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 2 to about 90% of theweight of a given unit dosage form. The amount of active compound insuch therapeutically useful compositions is such that an effectivedosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the active compound maybe incorporated into sustained-release preparations, particles, anddevices.

The active compound may also be administered intravenously orintramuscularly by infusion or injection. Solutions of the activecompound or its salts can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form should be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, the preferred methods of preparationare vacuum drying and the freeze drying techniques, which yield a powderof the active ingredient plus any additional desired ingredient presentin the previously sterile-filtered solutions.

For topical administration, the present compounds may be applied in pureform, i.e., when they are liquids. However, it will generally bedesirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina, nanoparticles, and thelike. Useful liquid carriers include water, alcohols or glycols orwater-alcohol/glycol blends, in which the present compounds can bedissolved or dispersed at effective levels, optionally with the aid ofnon-toxic surfactants. Adjuvants such as fragrances and additionalantimicrobial agents can be added to optimize the properties for a givenuse. The resultant liquid compositions can be applied from absorbentpads, used to impregnate bandages and other dressings, or sprayed ontothe affected area using pump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Useful dosages of the compounds of formula I can be determined bycomparing their in vitro activity, and in vivo activity in animalmodels. Methods for the extrapolation of effective dosages in mice, andother animals, to humans are known to the art; for example, see U.S.Pat. No. 4,938,949.

The amount of the compound, or an active salt or derivative thereof,required for use in treatment will vary not only with the particularsalt selected but also with the route of administration, the nature ofthe condition being treated and the age and condition of the patient andwill be ultimately at the discretion of the attendant physician orclinician.

In general, however, a suitable dose will be in the range of from about1 to about 500 mg/kg, e.g., from about 5 to about 400 mg/kg of bodyweight per day, such as 1 to about 250 mg per kilogram body weight ofthe recipient per day.

The compound is conveniently formulated in unit dosage form; forexample, containing 5 to 500 mg, 10 to 400 mg, or 5 to 100 mg of activeingredient per unit dosage form. In one embodiment, the inventionprovides a composition comprising a compound of the invention formulatedin such a unit dosage form.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations.

Co-administration of a compound disclosed herein with one or more otheractive therapeutic agents (e.g., antibacterial agents) generally refersto simultaneous or sequential administration of a compound disclosedherein and one or more other active therapeutic agents, such thattherapeutically effective amounts of disclosed herein and one or moreother active therapeutic agents are both present in the body of thepatient.

The ability of a compound disclosed herein to inhibit a bacterial effluxpump can be determined using a method as described in Example 12 and asshown in Table 1.

TABLE 1 *Enhanced **Enhanced Activity in Activity in E. coli P.aeruginosa μg/ml μg/ml Example Structure (fold increase) (fold increase)1

 12.5 (16x) 12.5 (2x) 2

 6.25 (32x)  6.25 (32x) 3

 3.13 (32x) 6.25 (4x) 4

6.25 (8x)  12.5 (32x) 5

3.13 (4x) 12.5 (1x) 6

3.13 (2x) 6.25 (1x) 7

3.13 (2x) 6.25 (1x) 8

3.13 (8x) 6.25 (2x) 9

 3.13 (64x)  6.25 (32x) 10

 6.25 (512x) 6.25 (1x) 11

 12.5 (16x)  12.5 (16x) 12

6.25 (4x) 6.25 (8x) 13

 6.25 (16x) 6.25 (8x) 14

12.5 (8x) 12.5 (1x) 15

 6.25 (256x) 3.13 (1x) 16

 12.5 (256x)   25 (2x) 17

3.13 (8x) 3.13 (1x) 18

3.13 (8x) 3.13 (1x) 19

3.13 (1x) 6.25 (2x) 20

6.25 (8x) 12.5 (1x) 21

 6.25 (64x)  12.5 (16x) 22

6.25 (8x) 6.25 (8x) 23

 6.25 (16x    6.25 (16x) 24

6.25 (4x) 12.5 (8x) 25

 6.25 (64x) 6.25 (4x   *These data were generated using clarithromycinas the antibiotic and the various EPIs against Escherichia coli ATCC25922. **These data were generated using levofloxacin as the antibioticand the various EPIs against Pseudomonas aeruginosa ATCC 27853.

The invention will now be illustrated by the following non-limitingexamples.

PREPARATION OF INTERMEDIATES

Table 2 shows amine intermediates that were used to prepare compoundsdescribed herein.

TABLE 2

Intermediate A

Intermediate B

Intermediate C

Intermediate D

Intermediate E

Intermediate F

Scheme 3 illustrates a general method for the preparation of certainamine intermediates.

The variables X and Y represent protecting groups as needed. Thevariable W represents a (C₂-C₁₃)alkyl corresponding to the R¹ variablefor compounds of formula I. It is to be understood that the two nitrogenatoms attached to W are attached on different carbon atoms of W.

Preparation of Amine Intermediate A (Di-tert-butyl(5-aminopentane-1,4-diyl)(R)-dicarbamate)

Di-tert-butyl (5-aminopentane-1,4-diyl)(R)-dicarbamate

Di-tert-butyl(5-(1,3-dioxoisoindolin-2-yl)pentane-1,4-diyl)(R)-dicarbamate (1.71 g,2.24 mmol) was dissolved in methanol (20 mL) and hydrazine monohydrate(220 μL, 4.47 mmol) was added. The reaction mixture was then refluxedfor 2 hours and cooled to room temperature. The precipitate formed wasfiltered and methanol used to wash the filtrate. The filtrate wasconcentrated under reduced pressure and the remaining oil purified usingan ISCO chromatograph with silica (0-10% methanol/methylene chloride+1%NH₄OH) to give product as a yellow oil. (560 mg, 79%); ¹H NMR (CDCl₃)(300 MHz) δ 4.62 (m, 2H), 3.52 (m, 1H), 3.14-3.09 (m, 2H), 2.79-2.60 (m,2H), 1.64-1.57 (m, 4H), 1.48-1.23 (m, 18H)

The requisite intermediates were prepared as shown in the followingsteps.

Step 1)

Di-tert-butyl (5-hydroxypentane-1,4-diyl)(R)-dicarbamate

To a solution of (R)-2,5-bis((tert-butoxycarbonyl)amino)pentanoic acid(1.70 g, 5.11 mmol) in THF 30 ml at −15° C. were successively added asolution of N-methyl morpholine (620 μL, 5.70 mmol) and isobutylchloroformate (668 μL, 5.11 mmol). The reaction was stirred at −15° C.to −10° C. for 15 minutes. The precipitated N-methyl morpholine HCl wasremoved by filtration and washed with THF (10 mL), the combine filtrateswere chilled to −15° C. in an ice-salt bath. Then a solution of sodiumborohydride (580 mg, 15.33 mmol) in water (4 mL) was added in oneportion at −15° C. This reaction mixture was stirred at this temperaturefor 10 minutes. The reaction was quenched by the addition of saturatedaq. NH₄Cl and the resulting mixture was extracted with ethyl acetate.The organic layer was washed with brine and dried over sodium sulfate.The solution was then filtered and concentrated under reduced pressure.The crude product was used directly for next step without furtherpurification.

Step 2)

Di-tert-butyl(5-(1,3-dioxoisoindolin-2-yl)pentane-1,4-diyl)(R)-dicarbamate

Triphenylphosphine (1.61 g, 6.13 mmol) and phthalimide (902 mg, 6.13mmol) were added to a flask containing dry THF (40 mL). Di-tert-butyl(5-hydroxypentane-1,4-diyl)(R)-dicarbamate (1.63 g, 5.11 mmol) was addedand the flask was cooled to 0° C. DIAD (1.24 g, 6.13 mmol) was addeddropwise and reaction allowed to stir for 30 minutes at 0° C. andovernight at room temperature. The mixture was concentrated underreduced pressure and residue purified using an ISCO chromatograph withsilica (0-100% ethyl acetate/hexane) to give product as a white solid.(1.71 g, 74%); ¹H NMR (CDCl₃) (300 MHz) δ 7.89-7.82 (m, 2H), 7.76-7.69(m, 2H), 4.64-4.62 (m, 2H), 3.97-3.94 (m, 1H) 3.74-3.67 (m, 2H),3.15-3.13 (m, 2H), 1.66-1.52 (m, 4H), 1.52-1.43 (m, 9H), 1.27-1.23 (m,9H).

Preparation of Amine Intermediate B (di-tert-butyl(5-aminopentane-1,4-diyl)(S)-dicarbamate)

Di-tert-butyl (5-aminopentane-1,4-diyl)(S)-dicarbamate

Di-tert-butyl(5-(1,3-dioxoisoindolin-2-yl)pentane-1,4-diyl)(S)-dicarbamate (760 mg,1.70 mmol) formed was dissolved in methanol (30 mL) and hydrazinemonohydrate (177 μL, 3.40 mmol) was added. The reaction mixture was thenrefluxed for 2 hours and cooled to room temperature. The precipitateformed was filtered and methanol used to wash the filtrate. The filtratewas concentrated under reduced pressure and the remaining oil purifiedusing an ISCO column chromatography on silica gel (0-10%methanol/methylene chloride with 1% NH₃H₂O) to give product as a yellowoil. (450 mg, 83% yield); ¹H NMR (CDCl₃) (300 MHz) δ 4.63 (m, 2H),3.52-3.49 (m, 1H), 3.14-3.12 (m, 2H), 2.79-2.60 (m, 2H), 1.54-1.57 (m,4H), 1.53-1.26 (m, 18H).

The requisite intermediates were prepared as shown in the followingsteps.

Step 1)

Di-tert-butyl (5-hydroxypentane-1,4-diyl)(S)-dicarbamate

To a solution of (S)-2,5-bis((tert-butoxycarbonyl)amino)pentanoic acid(1000 mg, 3.01 mmol) in THF 30 mL at −15° C. were successively added asolution of N-methyl morpholine (305 μL, 3.32 mmol) and isobutylchloroformate (411 μL, 3.01 mmol). The reaction was stirred at −15° C.to −10° C. for 15 minutes. The precipitated N-methyl morpholine HCl wasremoved by filtration and washed with THF (10 mL), the combine filtrateswere chilled to −15° C. in an ice-salt bath. Then a solution of sodiumborohydride (342 mg, 9.03 mmol) in water (4 mL) was added in one portionat −15° C. This reaction mixture was stirred at this temperature for 10minutes. The reaction was quenched by the addition of saturated aq.NH₄Cl and the resulting mixture was extracted with ethyl acetate. Theorganic layer was washed with brine and dried over sodium sulfate. Thesolution was then filtered and concentrated under reduced pressure, andthe residue purified on column (0-100% ethyl acetate/hexane) to giveproduct as a white powder (750 mg, 78% yield); ¹H NMR (CDCl₃) (300 MHz)δ 4.74-4.64 (m, 2H), 3.63-3.55 (m, 3H), 3.14-3.13 (m, 2H), 2.45 (m, 1H),1.68-1.58 (m, 4H), 1.56-1.44 (m, 18H).

Step 2)

Di-tert-butyl(5-(1,3-dioxoisoindolin-2-yl)pentane-1,4-diyl)(S)-dicarbamate

Triphenylphosphine (742 mg, 2.83 mmol) and phthalimide (417 mg, 2.83mmol) were added to a flask containing dry THF (15 mL). Di-tert-butyl(5-hydroxypentane-1,4-diyl)(S)-dicarbamate (750 mg, 2.36 mmol) was addedand the flask was cooled to 0° C. DIAD (573 mg, 2.83 mmol) was addeddropwise and reaction allowed to stir for 30 minutes at 0° C. andovernight at room temperature. The mixture was concentrated underreduced pressure and residue was purified using an ISCO chromatographywith silica gel (0-100% ethyl acetate/hexane) to give product as a whitesolid. (760 mg, 72% yield); ¹H NMR (CDCl₃) (300 MHz) δ 7.86-7.83 (m,2H), 7.72-7.69 (m, 2H), 4.64-4.61 (m, 2H), 3.97-3.94 (m, 1H) 3.70-3.67(m, 2H), 3.15-3.13 (m, 2H), 1.67-1.54 (m, 4H), 1.52-1.37 (m, 9H),1.37-1.22 (m, 9H).

Preparation of Amine Intermediate C.

Intermediate C was prepared using procedures similar to those used toprepare intermediates A, B, D, E, and F.

Preparation of amine intermediate D (Di-tert-butyl((2S)-1-aminohexane-2,5-diyl)dicarbamate)

Di-tert-butyl ((2S)-1-aminohexane-2,5-diyl)dicarbamate

The di-tert-butyl((2S)-1-(1,3-dioxoisoindolin-2-yl)hexane-2,5-diyl)dicarbamate (320 mg,0.694 mmol) was dissolved in methanol (20 mL) and hydrazine monohydrate(0.136 mL, 2.78 mmol) was added. The reaction mixture was then refluxedfor 2 hours and cooled to room temperature. The precipitate formed wasfiltered and methanol used to wash the filtrate. The filtrate wasconcentrated under reduced pressure. The reaction was diluted withEtOAc, washed with saturated NaHCO₃, saturated ammonium chloride andbrine sequentially. The organic layer was dried over sodium sulfate andconcentrated under reduced pressure. The crude product was used directlywithout further purification; ¹H NMR (CDCl₃) (300 MHz) δ 4.98-4.90 (m,1H), 4.72-4.41 (m. 1H), 3.59-3.48 (m, 1H), 2.75-2.56 (m, 1H), 1.48-1.021(m, 25H).

The requisite intermediates were prepared as shown in the followingsteps.

Step 1)

(S)-tert-Butyl(1-((tert-butyldiphenylsilyl)oxy)-5-oxohexan-2-yl)carbamate

To a solution(S)-tert-butyl-2-(((tert-butyldiphenylsilyl)oxyl)methyl)-5-oxopyrrolidin-1-carboxylate(1.8 g, 3.97 mmol) in THF (400 mL) at −78° C. was added 3 M methylmagnesium chloride (1.45 mL, 4.37 mmol) dropwise. After the mixture wasstirred at −78° C. for 2 hours, the reaction was warmed up to 0° C. andstirred at room temperature for another 2 hours. The reaction mixturewas diluted with DCM, washed with saturated NaHCO₃ and brine, dried oversodium sulfate and concentrated under reduced pressure, poured intowater and extracted with ethyl acetate. The residue was purified on ISCO(0-70% ethyl acetate/hexane to give product as a white solid. (1.5 g,78%); ¹H NMR (CDCl₃) (300 MHz) δ 7.72 (m, 4H), 7.50 (m, 6H), 4.78 (m,1H), 3.75-3.69 (m, 3H), 2.55 (m, 2H), 2.15 (s, 3H), 1.95 (m, 2H), 1.53(s, 9H), 1.16 (s, 9H);

Step 2)

tert-Butyl((2S)-5-amino-1-((tert-butyldiphenylsilyl)oxy)hexan-2-yl)carbamate

To a solution (S)-tert-butyl(1-((tert-butyldiphenylsilyl)oxy)-5-oxohexan-2-yl)carbamate (0.70 mg,9.03 mmol) and ammonium acetate (2.01 g, 26.1 mmol) in MeOH (100 mL) wasadded molecular sieve and sodium cyanoborohydride (1.64 g, 26.1 mmol).The reaction was stirred at room temperature overnight. The molecularsieve was filtered off and washed with EtOAc. The filtrate wasconcentrated under reduced pressure and partitioned between EtOAc andsat. NaHCO₃ solution, extracted with EtOAc three time. The combinedorganic layer was washed with brine, concentrated under reducedpressure. The crude product was used directly in the next step withoutfurther purification.

Step 3)

Di-tert-butyl((2S)-1-((tert-butyldiphenylsilyl)oxy)hexane-2,5-diyl)dicarbamate

To a solution tert-butyl((2S)-5-amino-1-((tert-butyldiphenylsilyl)oxy)hexan-2-yl)carbamate (0.65g, 1.34 mmol) in DCM (25 mL) at room temperature was added DIPEA (0.47mL, 2.68 mmol) and (Boc)₂O (0.584 g, 2.68 mmol). The reaction wasstirred at room temperature overnight. The reaction mixture was dilutedwith DCM, washed with saturated NaHCO₃ and brine, dried over sodiumsulfate and concentrated under reduced pressure. The residue waspurified on ISCO (0-100% ethyl acetate/hexane) to give product as awhite solid. (586 mg, 75%); ¹H NMR (CDCl₃) (300 MHz) δ 7.74 (m, 5H),7.50 (m, 5H), 4.78 (m, 1H), 4.40 (m. 1H), 3.69 (m, 5H), 1.54-1.33 (m,31H), 1.12 (m, 3H);

Step 4)

Di-tert-butyl ((2S)-1-hydroxyhexane-2,5-diyl)dicarbamate

To a solution di-tert-butyl((2S)-1-((tert-butyldiphenylsilyl)oxy)hexane-2,5-diyl)dicarbamate (0.50g, 0.86 mmol) in THF (15 mL) at 0° C. was added 1 M TBAF (3.43 mL, 3.43mmol). The reaction was stirred at room temperature overnight. Thereaction mixture was quenched with saturated ammonium chloride,extracted with EtOAc three times. The organic layer was washed withbrine, dried over sodium sulfate and concentrated under reducedpressure. The residue was purified on ISCO (0-70% ethyl acetate/hexane)to give product as a white solid. (290 mg, 97%). ¹H NMR (CDCl₃) (300MHz) δ 4.96-4.86 (m, 1H), 4.55-4.36 (m. 1H), 3.57 (m, 3H), 3.13 (m, 1H),1.61-1.22 (m, 22H), 1.08 (m, 3H);

Step 5)

Di-tert-butyl((2S)-1-(1,3-dioxoisoindolin-2-yl)hexane-2,5-diyl)dicarbamate

Triphenylphosphine (237 mg, 0.905 mmol) and phthalimide (133 mg, 0.905mmol) were added to a flask containing dry THF (50 mL). Di-tert-butyl((2S)-1-hydroxyhexane-2,5-diyl)dicarbamate (250 mg, 0.754 mmol) wasadded and the flask was cooled to 0° C. DIAD (183 mg, 0.905 mmol) wasadded dropwise and reaction allowed to stir for 30 minutes at 0° C. andovernight at room temperature. The mixture was concentrated underreduced pressure and residue purified using an ISCO chromatograph (0-70%ethyl acetate/hexane) to give product (320 mg) as a white solid ¹H NMR(CDCl₃) (300 MHz) δ 7.82 (m, 2H), 7.69 (m, 2H), 4.70-4.58 (m, 1H), 4.37(m. 1H), 3.92 (m, 1H), 3.65 (m, 3H), 1.61-1.11 (m, 26H);

Step 6)

Di-tert-butyl ((2S)-1-aminohexane-2,5-diyl)dicarbamate

The Di-tert-butyl((2S)-1-(1,3-dioxoisoindolin-2-yl)hexane-2,5-diyl)dicarbamate (320 mg,0.694 mmol) was dissolved in methanol (20 mL) and hydrazine monohydrate(0.136 mL, 2.78 mmol) was added. The reaction mixture was then refluxedfor 2 hours and cooled to room temperature. The precipitate formed wasfiltered and methanol used to wash the filtrate. The filtrate wasconcentrated under reduced pressure. The reaction was diluted withEtOAc, washed with saturated NaHCO₃, saturated ammonium chloride andbrine sequentially. The organic layer was dried over sodium sulfate andconcentrated under reduced pressure. The crude product was used directlywithout further purification; ¹H NMR (CDCl₃) (300 MHz) δ 4.98-4.90 (m,1H), 4.72-4.41 (m. 1H), 3.59-3.48 (m, 1H), 2.75-2.56 (m, 1H), 1.48-1.021(m, 25H).

Preparation of amine intermediates E and F (tert-butyl(((3S,5R)-5-(aminomethyl)-1-benzylpyrrolidin-3-yl)methyl)carbamate andtert-butyl(((3R,5S)-5-(aminomethyl)-1-benzylpyrrolidin-3-yl)methyl)carbamate)

tert-Butyl(((3S,5R)-5-(aminomethyl)-1-benzylpyrrolidin-3-yl)methyl)carbamate

tert-Butyl(((3R,5S)-5-(aminomethyl)-1-benzylpyrrolidin-3-yl)methyl)carbamate

To a solution of tert-butyl(((5S)-1-benzyl-5-(hydroxymethyl)pyrrolidin-3-yl)methyl)carbamate (1.58g, 5.23 mmol), triphenylphosphine (1.51 g, 5.75 mmol) and phthalimide(846 mg, 5.75 mmol) in THF (20 mL) was added DIAD (1.16 mL, 5.75 mmol)at 0° C. It was stirred at 0° C.—room temperature and monitored by TLC.After finishing the reaction it was concentrated under reduced pressureand the residue purified on column chromatography with silica gel using50-90% ethyl acetate in hexanes to give crude product as an off whitesolid (2.8 g, ˜80% purity).

To the solution of the above crude product (2.8 g, ˜80% purity, ˜5.2mmol) in MeOH (30 mL) was added hydrazine monohydrate (1.8 mL, 36.0mmol). The mixture was stirred at 80° C. for 1 h then cooled to roomtemperature. The solvent was removed and the residue was triturated withCH₂Cl₂. The white solid was removed by filtration and the filtrate wasconcentrated under reduced pressure and the residue purified by columnchromatography on silica gel. Elution with EtOAc then 1% NH₃.H₂O in 10%MeOH/CH₂Cl₂ afforded the top spot (386 mg, yellow oil, 25% yield in 2steps) as tert-butyl (((3S,5R)-5-(aminomethyl)-1-benzylpyrrolidin-3-yl)methyl)carbamate. ¹H NMR(300 MHz, CDCl₃) δ 7.30 (m, 5H), 5.28 (br. S, 1H), 3.99 (d, J=13.5 Hz,1H), 3.15 (d, J=12.9 Hz, 1H), 3.09 (m, 2H), 2.70-2.90 (m, 3H), 2.51 (m,1H), 2.04-2.34 (m, 3H), 1.36-1.50 (m, 10H), and the bottom spot (498 mg,white solid, 32% yield in 2 steps) as tert-butyl (((3S,5R)-5-(aminomethyl)-1-benzylpyrrolidin-3-yl)methyl)carbamate. ¹H NMR(300 MHz, CDCl₃) δ 7.30 (m, 5H), 4.52 (br. S, 1H), 3.57 (d, J=12.9 Hz,1H), 3.47 (d, J=12.9 Hz, 1H), 2.81-3.02 (m, 5H), 1.80-1.95 (m, 2H), 1.59(m, 1H), 1.43 (s, 9H), 0.70 (m, 1H)

The requisite intermediates were prepared as shown in the followingsteps.

Step 1)

tert-Butyl(((5S)-1-benzyl-5-(hydroxymethyl)pyrrolidin-3-yl)methyl)carbamate

To a solution of (2S)-methyl 1-benzyl-4-cyanopyrrolidine-2-carboxylate(2.37 g mg, 9.72 mmol) in dry THF (50 mL) at 0° C. under N₂ was addedLAH (730 mg, 19.4 mmol) in several portions. The reaction mixture wasstirred at 0° C. for 30 minutes then room temperature for 1 h. Then thereaction mixture was cooled to 0° C. and slowly added H₂O (0.7 mL), 15%NaOH solution (0.7 mL), EtOAc, and H₂O (2.8 mL). After stirring at roomtemperature for 30 min Na₂SO₄ was added. Continued to stir for 30minutes then the solid was removed by passing a Celite pad. The filtratewas concentrated to give a crude intermediate((2S)-4-(aminomethyl)-1-benzylpyrrolidin-2-yl)methanol. The crudeintermediate was not further purified and identified. It was directlyused in next step. The above intermediate was dissolved in DCM (30 mL)then it was added Boc₂O (2.54 g, 11.7 mmol) and TEA (2.02 mL, 14.6mmol). The reaction mixture was stirred at room temperature overnight.The reaction mixture was diluted with DCM and washed with water, brinethen dried over anhydrous sodium sulfate. The solvent was removed andthe residue was purified by column chromatography on silica gel usingEtOAc. The desired product was collected (1.58 g, 54% yield) as lightyellow oil. ¹H NMR (300 MHz, CDCl₃) δ 7.30 (m, 5H), 4.84 (br. S, 1H),3.98 (d, J=13.5 Hz, 1H), 3.72 (m, 1H), 3.48 (d, J=11.1 Hz, 1H), 3.26 (d,J=12.9 Hz, 1H), 3.07 (m, 2H), 2.73 (m, 2H), 2.43 (m, 1H), 2.26 (m, 1H),2.10 (m, 1H), 1.60 (m, 2H), 1.42 (s, 9H).

Example 1. Preparation of(R)—N-(2,5-diaminopentyl)-3,6-bis(4-fluorophenyl)-1H-indole-2-carboxamidedihydrochloride

(R)—N-(2,5-Diaminopentyl)-3,6-bis(4-fluorophenyl)-1H-indole-2-carboxamidedihydrochloride

To a solution of di-tert-butyl(5-(3,6-bis(4-fluorophenyl)-1H-indole-2-carboxamido)pentane-1,4-diyl)(R)-dicarbamate(16 mg, 0.03 mmol) in MeOH (5 mL) was added HCl solution (4 M indioxane, 0.1 mL, 0.4 mmol). It was stirred at r.t. until no startingmaterial left. Then the solvent was removed under vacuo. The residue wastriturated with EtOAc and the precipitate was collected as an off-whitepowder (18 mg, 65% yield). ¹H NMR (300 MHz, D₂O) δ 7.68 (m, 3H), 7.56(m, 3H), 7.34 (m, 3H), 7.23 (m, 2H), 3.61 (m, 1H), 3.53 (m, 1H), 3.48(m, 1H), 3.03 (m, 2H), 1.77 (m, 2H), 1.69 (m, 2H). MS: Calcd forC₂₆H₂₆F₂N₄O 449.21 [M+H]⁺, found 449.25 [M+H]⁺.

The requisite intermediates were prepared as shown in the followingsteps.

Step 1)

methyl 3,6-dibromo-1H-indole-2-carboxylate

To a solution of methyl 6-bromo-1H-indole-2-carboxylate (1.02 g, 4 mmol)in dry THF (25 mL) was added NBS (783 mg, 4.4 mmol). The reactionmixture was heated at 70° C. until TLC showed no starting material left.Then it was cooled to room temperature and concentrated under reducedpressure. The formed crystal was filtered off and washed with THF togive the product (1.09 g, 82% yield) as a white crystal. It was used fornext step reaction without further purification. MS: Calcd forC₁₀H₇Br₂NO₂ 331.88 [M−H]⁻, found 331.75 [M−H]⁻.

Step 2)

methyl 3,6-bis(4-fluorophenyl)-1H-indole-2-carboxylate

The mixture of methyl 3,6-dibromo-1H-indole-2-carboxylate (890 mg, 2.5mmol), (4-fluorophenyl)boronic acid (1.22 g, 8.75 mmol) in toluene (30mL), EtOH (6 mL) and saturated NaHCO₃ solution (6 mL) was degassed andPd(dppf)Cl₂ (200 mg, 0.25 mmol) was added. The mixture was heated at105° C. overnight and it was extracted with EtOAc. The organic phaseswere combined, washed with brine, concentrated under reduced pressureand the residue was purified by column chromatography on silica gel(40-60% ethyl acetate/hexanes) to give the product (800 mg, 88% yield)as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 8.98 (br, 1H), 7.63 (m, 3H),7.54 (m, 2H), 7.37 (d, J=8.4 Hz, 2H), 7.15 (m, 4H), 3.84 (s, 3H). MS:Calcd for C₂₂H₁₅F₂NO₂ 364.11 [M+H]⁺, found 364.15 [M+H]⁺.

Step 3)

3,6-Bis(4-fluorophenyl)-1H-indole-2-carboxylic Acid

To a solution of methyl 3,6-bis(4-fluorophenyl)-1H-indole-2-carboxylate(0.80 g, 2.2 mmol) in MeOH (10 mL) was added NaOH solution (2 M, 10 mL).The reaction mixture was stirred at room temperature until no startingmaterial left. Organic solvent was removed under reduced pressure andthe residue was acidified with HCl solution. The precipitate wasfiltered and washed with water. The solid was dried to provide theproduct as an off-white powder (600 mg, 86% yield) which was used fornext step reaction without further purification. ¹H NMR (300 MHz,DMSO-d₆) δ 11.60 (br, 1H), 7.66 (m, 5H), 7.57 (m, 2H), 7.27 (m, 4H). MS:Calcd 15 for C₂₁H₁₃F₂NO₂, found 348.09 [M−H]⁻ found 697.20 [2M−H]⁻.

Step 4)

Di-tert-butyl(5-(3,6-bis(4-fluorophenyl)-1H-indole-2-carboxamido)pentane-1,4-diyl)(R)-dicarbamate

To a solution of 3,6-bis(4-fluorophenyl)-1H-indole-2-carboxylic acid (40mg, 0.11 mmol) in anhydrous DMF (1 mL) was added DIPEA (0.05 mL, 0.3mmol), HOBt (11 mg, 0.07 mmol) and EDC (25 mg, 0.13 mmol). The reactionmixture was stirred at room temperature and di-tert-butyl(5-aminopentane-1,4-diyl)(R)-dicarbamate (intermediate A) (36 mg, 0.11mmol) was added. The reaction mixture was continued to stir at roomtemperature overnight. The reaction mixture was then extracted withEtOAc and washed with water and brine. The organic layer was dried overanhydrous sodium sulfate and filtered. The filtrate was concentratedunder reduced pressure and the residue purified by column chromatographyon silica gel (40-60% ethyl acetate/hexanes) to give the product (35 mg,47% yield) as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 9.35 (br, 1H),7.59 (m, 3H), 7.51 (m, 2H), 7.45 (m, 2H), 7.32 (m, 2H), 7.14 (t, J=8.7Hz, 2H), 6.03 (br, 1H), 4.62 (br, 1H), 4.47 (br, 1H), 3.51 (m, 2H), 3.35(m, 1H), 3.09 (m, 2H), 1.58 (m, 4H), 1.42 (s, 9H), 1.39 (s, 9H). MS:Calcd for C₃₆H₄₂F₂N₄O₅ 647.35 [M−H]⁻, found 647.35 [M−H]⁻.

Example 2. Preparation of(S)-3-cyclopropyl-N-(2,5-diaminopentyl)-6-(4-fluorophenyl)-1H-indole-2-carboxamidedihydrochloride

(S)-3-cyclopropyl-N-(2,5-diaminopentyl)-6-(4-fluorophenyl)-1H-indole-2-carboxamidedihydrochloride

To a solution of di-tert-butyl(5-(3-cyclopropyl-6-(4-fluorophenyl)-1H-indole-2-carboxamido)pentane-1,4-diyl)(S)-dicarbamate(41 mg, 0.07 mmol) in MeOH (5 mL) was added HCl in dioxane (4 M, 0.15mL, 0.6 mmol). The reaction mixture was stirred at room temperatureuntil TLC showed no starting material left. It was concentrated underreduced pressure and triturated with EtOAc to afford the crude productwhich was purified by C18 reversed column chromatography usingMeOH/water (0-50%). A white powder was collected after removing thesolvent under reduced pressure (12 mg, 37% yield). ¹H NMR (300 MHz,CD₃OD) δ 7.83 (d, J=8.4 Hz, 1H), 7.66 (m, 3H), 7.33 (d, J=8.4 Hz, 1H),7.17 (d, J=8.4 Hz, 2H), 3.81 (m, 1H), 3.76 (m, 1H), 3.54 (m, 1H), 3.03(m, 2H), 2.22 (m, 1H), 1.83 (m, 4H), 1.19 (m, 2H), 0.85 (m, 2H). MS:Calcd for C₂₃H₂₇FN₄O 393.22 [M−H]⁻, found 393.15 [M−H]⁻.

The requisite intermediates were prepared as shown in the followingsteps.

Step 1)

1-(tert-Butyl) 2-methyl3-bromo-6-(4-fluorophenyl)-1H-indole-1,2-dicarboxylate

To a solution of methyl3-bromo-6-(4-fluorophenyl)-1H-indole-2-carboxylate (348 mg, 1 mmol) inDCM (15 mL) was added Boc₂O (330 mg, 1.5 mmol) and DMAP (24 mg, 0.2mmol). It was stirred at room temperature overnight and TLC showed nostarting material left. It was extracted with EtOAc and washed withNH₄Cl solution and brine. After concentration, the white powder was usedfor next step reaction without further purification (0.42 g, 94%). ¹HNMR (300 MHz, CDCl₃) δ 8.35 (m, 1H), 7.64 (m, 3H), 7.60 (m, 1H), 7.16(t, J=8.7 Hz, 2H), 4.00 (s, 3H), 1.63 (s, 9H). MS (ESI−): Calcd forC₂₁H₁₉BrFNO₄ 446.05 [M−H]⁻, found 491.00 [M+HCOOH−H]⁻.

Step 2)

1-(tert-Butyl) 2-methyl3-cyclopropyl-6-(4-fluorophenyl)-1H-indole-1,2-dicarboxylate

1-(tert-Butyl) 2-methyl3-bromo-6-(4-fluorophenyl)-1H-indole-1,2-dicarboxylate (225 mg, 0.5mmol) and cyclopropylboronic acid (86 mg, 1 mmol) in a mixture oftoluene, ethanol and sat. Na₂CO₃ solution (10/2/2 mL) was degassed andPd(dppf)Cl₂ (35 mg, 0.04 mmol) was added. The reaction mixture washeated at 105° C. overnight and it was extracted with EtOAc and washedwith brine and concentrated under reduced pressure. The residue was thenpurified by column chromatography on silica gel (0-10% ethylacetate/hexanes) to give the product (45 mg, 22% yield) as an off-whitepowder. ¹H NMR (300 MHz, CDCl₃) δ 7.65 (m, 4H), 7.42 (m, 1H), 7.14 (m,2H), 3.96 (s, 3H), 2.06 (m, 1H), 1.60 (s, 9H), 0.95 (m, 2H), 0.90 (m,2H).

Step 3)

3-Cyclopropyl-6-(4-fluorophenyl)-1H-indole-2-carboxylic Acid

To a solution of 1-(tert-butyl) 2-methyl3-cyclopropyl-6-(4-fluorophenyl)-1H-indole-1,2-dicarboxylate (44 mg,0.11 mmol) in THF (3 mL) was added NaOH solution (2 M, 3 mL). Thereaction mixture was heated at 70° C. until no starting material left.THF was removed under reduced pressure and the residue was acidifiedwith HCl solution. The precipitate was filtered and washed with water.The solid was dried to provide the product as an off-white powder (30mg, 92% yield) which was used for next step reaction without furtherpurification. MS: Calcd for C₁₈H₁₄FNO₂ 294.10 [M−H]⁻, found 294.05[M−H]⁻.

Step 4)

Di-tert-butyl(5-(3-cyclopropyl-6-(4-fluorophenyl)-1H-indole-2-carboxamido)pentane-1,4-diyl)(S)-dicarbamate

To a solution of 3-cyclopropyl-6-(4-fluorophenyl)-1H-indole-2-carboxylicacid (30 mg, 0.09 mmol) in dry DMF (1 mL) was added DIPEA (0.05 mL, 0.3mmol), HOBt (16 mg, 0.1 mmol) and EDC (38 mg, 0.2 mmol). The reactionmixture was stirred at room temperature and di-tert-butyl(5-aminopentane-1,4-diyl)(S)-dicarbamate (intermediate B) (32 mg, 0.1mmol) was added. The reaction mixture was stirred at room temperatureovernight. It was diluted and extracted with EtOAc and washed with waterand brine. The combined organic layer was dried over anhydrous sodiumsulfate and filtered. The filtrate was concentrated under reducedpressure and the residue purified by column chromatography on silica gelusing 40-45% EtOAc in hexane to give the product (41 mg, 73% yield) as apale white powder. MS: Calcd for C₃₃H₄₃FN₄O₅ 595.32 [M+H]⁺, found 595.40[M+H]⁺.

Example 3. Preparation of(S)-3-benzyl-N-(2,5-diaminopentyl)-6-(4-fluorophenyl)-1H-indole-2-carboxamidedihydrochloride

(S)-3-Benzyl-N-(2,5-diaminopentyl)-6-(4-fluorophenyl)-1H-indole-2-carboxamidedihydrochloride

To a solution of di-tert-butyl(5-(3-benzyl-6-(4-fluorophenyl)-1H-indole-2-carboxamido)pentane-1,4-diyl)(S)-dicarbamate(26 mg, 0.04 mmol) in MeOH (3 mL) was added HCl solution (4 M indioxane, 0.15 mL, 0.6 mmol). The reaction mixture was stirred at roomtemperature and after the reaction was complete the solvent was removedunder reduced pressure. The residue was triturated with EtOAc and theprecipitate was collected as an off-white powder (12 mg, 56% yield). ¹HNMR (300 MHz, CD₃OD) δ 7.60 (m, 3H), 7.50 (d, J=8.4 Hz, 1H), 7.27 (m,3H), 7.16 (m, 2H), 7.10 (m, 3H), 4.50 (s, 2H), 3.67 (m, 1H), 3.61 (m,1H), 3.37 (m, 1H), 2.91 (m, 2H), 1.81 (m, 4H). MS: Calcd for C₂₇H₂₉FN₄O443.23 [M−H]⁻, found 443.25 [M−H]⁻.

The requisite intermediates were prepared as shown in the followingsteps.

Step 1)

1-(tert-Butyl) 2-methyl3-benzyl-6-(4-fluorophenyl)-1H-indole-1,2-dicarboxylate

1-(tert-Butyl) 2-methyl3-bromo-6-(4-fluorophenyl)-1H-indole-1,2-dicarboxylate (270 mg, 0.6mmol) and 2-benzyl-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (160 mg, 0.72mmol) in a mixture of toluene, ethanol and sat. Na₂CO₃ solution (10/2/2mL) was degassed and Pd(dppf)Cl₂ (70 mg, 0.08 mmol) was added. Thereaction mixture was heated at 105° C. overnight and it was extractedwith EtOAc. The organic layer was washed with brine and concentratedunder reduced pressure. The residue was purified by columnchromatography on silica gel (0-15% ethyl acetate/hexanes) to give theproduct (50 mg, 18% yield) as an off-white powder. ¹H NMR (300 MHz,CDCl₃) δ 7.58 (m, 3H), 7.48 (m, 1H), 7.23 (m, 5H), 7.14 (m, 3H), 4.21(s, 2H), 3.91 (s, 3H), 1.54 (s, 9H).

Step 2)

3-Benzyl-6-(4-fluorophenyl)-1H-indole-2-carboxylic Acid

To a solution of 1-(tert-butyl) 2-methyl3-benzyl-6-(4-fluorophenyl)-1H-indole-1,2-dicarboxylate (50 mg, 0.11mmol) in THF (5 mL) was added NaOH solution (2 M, 3 mL). The reactionmixture was heated at 70° C. until no starting material left. THF wasremoved under reduced pressure and the residue was acidified with HClsolution. The precipitate was filtered and washed with water. It wasdried to provide the product as an off-white powder (34 mg, 89% yield)which was used for next step reaction without further purification. ¹HNMR (300 MHz, DMSO-d₆) δ 7.65 (m, 3H), 7.55 (m, 1H), 7.26 (m, 2H), 7.21(m, 6H), 4.50 (s, 2H). MS: Calcd for C₂₂H₁₆FNO₂ 344.12 [M−H]⁻, found344.00 [M−H]⁻.

Step 3)

di-tert-Butyl(5-(3-benzyl-6-(4-fluorophenyl)-1H-indole-2-carboxamido)pentane-1,4-diyl)(S)-dicarbamate

To a solution of 3-benzyl-6-(4-fluorophenyl)-1H-indole-2-carboxylic acid(30 mg, 0.09 mmol) in dry DMF (1 mL) was added DIPEA (0.05 mL, 0.3mmol), HOBt (16 mg, 0.1 mmol) and EDC (38 mg, 0.2 mmol). The reactionmixture was stirred at room temperature and di-tert-butyl(5-aminopentane-1,4-diyl)(S)-dicarbamate (intermediate B) (32 mg, 0.1mmol) was added. The reaction mixture was stirred at room temperatureovernight. It was diluted with EtOAc and washed with water and brine.The organic layer was dried over anhydrous sodium sulfate and filtered.The filtrate was concentrated under reduced pressure and the residuepurified by column chromatography on silica gel using 20-40% EtOAc inhexane to give the product (26 mg, 45% yield) as a pale-yellow powder.¹H NMR (300 MHz, CDCl₃) δ 9.28 (br, 1H), 7.65 (d, J=8.4 Hz, 2H), 7.58(m, 3H), 7.31 (m, 5H), 7.14 (t, J=8.7 Hz, 2H), 6.35 (br, 1H), 4.62 (br,2H), 4.44 (s, 2H), 3.63 (m, 1H), 3.46 (m, 1H), 3.39 (m, 1H), 3.09 (m,2H), 1.58 (m, 4H), 1.43 (s, 9H), 1.41 (s, 9H). MS: Calcd for C₃₇H₄₅FN₄O₅643.34 [M−H]⁻, found 643.40 [M−H]⁻.

Example 4. Preparation of(S)—N-(2,5-diaminopentyl)-6-(4-fluorophenyl)-3-(pyridin-4-yl)-1H-indole-2-carboxamidetrihydrochloride

(S)—N-(2,5-Diaminopentyl)-6-(4-fluorophenyl)-3-(pyridin-4-yl)-1H-indole-2-carboxamide

To a solution of di-tert-butyl(5-(6-(4-fluorophenyl)-3-(pyridin-4-yl)-1H-indole-2-carboxamido)pentane-1,4-diyl)(S)-dicarbamate(26 mg, 0.04 mmol) in MeOH (3 mL) was added HCl solution (4 M indioxane, 0.15 mL, 0.6 mmol). The reaction mixture was stirred at roomtemperature overnight and solvent was removed under reduced pressure.The residue was triturated with EtOAc and the precipitate was collectedas an off-white powder (20 mg, 72% yield). ¹H NMR (300 MHz, D₂O) δ 8.73(m, 2H), 8.27 (m, 3H), 7.91 (m, 1H), 7.76 (m, 2H), 7.28 (m, 3H), 3.64(m, 1H), 3.59 (m, 1H), 3.34 (m, 1H), 3.02 (m, 2H), 1.82 (m, 4H). MS:Calcd for C₂₅H₂₆FN₅O 430.21 [M−H]⁻, found 430.25 [M−H]⁻.

The requisite intermediates were prepared as shown in the followingsteps.

Step 1)

Methyl 6-(4-fluorophenyl)-3-(pyridin-4-yl)-1H-indole-2-carboxylate

The mixture of 1-(tert-butyl) 2-methyl3-bromo-6-(4-fluorophenyl)-1H-indole-1,2-dicarboxylate (270 mg, 0.6mmol), pyridin-4-ylboronic acid (110 mg, 0.9 mmol) in a mixture oftoluene, ethanol and sat. Na₂CO₃ solution (10/2/2 mL) was degassed andPd(dppf)Cl₂ (70 mg, 0.08 mmol) was added. The reaction mixture washeated at 110° C. overnight and it was extracted with EtOAc and washedwith brine and concentrated under reduced pressure. The residue was thenpurified by column chromatography on silica gel (0-30% MeOH/ethylacetate) to give the product (120 mg, 58% yield) as an off-white powder.¹H NMR (300 MHz, CDCl₃) δ 9.26 (br, 1H), 8.80 (m, 2H), 7.60 (m, 4H),7.48 (m, 3H), 7.17 (d, J=8.7 Hz, 2H), 3.86 (s, 3H). MS: Calcd forC₂₁H₁₅FN₂O₂ 345.11 [M−H]⁻, found 345.10 [M−H]⁻.

Step 2)

6-(4-Fluorophenyl)-3-(pyridin-4-yl)-1H-indole-2-carboxylic Acid

To a solution of 1-(tert-butyl) 2-methyl3-bromo-6-(4-fluorophenyl)-1H-indole-1,2-dicarboxylate (120 mg, 0.34mmol) in THF (5 mL) was added NaOH solution (2 M, 5 mL). The reactionmixture was heated at 70° C. until no starting material left. THF wasremoved under reduced pressure and the residue was acidified with HClsolution to pH about 5-6. The precipitate was filtered and washed withwater. It was dried to provide the product as an off-white powder (108mg, 94% yield), which was used for next step reaction without furtherpurification. ¹H NMR (300 MHz, DMSO-d₆) δ 8.57 (m, 2H), 7.65 (m, 2H),7.59 (m, 3H), 7.51 (m, 2H), 7.32 (m, 2H). MS: Calcd for C₂₀H₁₃FN₂O₂331.510 [M−H]⁻, found 331.05 [M−H]⁻.

Step 3)

di-tert-Butyl(5-(6-(4-fluorophenyl)-3-(pyridin-4-yl)-1H-indole-2-carboxamido)pentane-1,4-diyl)(S)-dicarbamate

To a solution of6-(4-fluorophenyl)-3-(pyridin-4-yl)-1H-indole-2-carboxylic acid (50 mg,0.15 mmol) in dry DMF (1.5 mL) was added DIPEA (0.05 mL, 0.3 mmol), HOBt(16 mg, 0.1 mmol) and EDC (35 mg, 0.18 mmol). The reaction mixture wasstirred at room temperature and di-tert-butyl(5-aminopentane-1,4-diyl)(S)-dicarbamate (intermediate B) (57 mg, 0.18mmol) was added. The reaction mixture was stirred at room temperatureovernight. It was extracted with EtOAc and washed with water and brine.The organic layer was dried over anhydrous sodium sulfate and filtered.The filtrate was concentrated under reduced pressure and the residuepurified by column chromatography on silica gel using 50-100% EtOAc inhexane to give the product (36 mg, 38% yield) as a pale brown powder. ¹HNMR (300 MHz, CDCl₃) δ 9.46 (br, 1H), 8.88 (d, J=6.0 Hz, 2H), 7.61 (m,1H), 7.58 (m, 3H), 7.52 (d, J=8.4 Hz, 1H), 7.35 (m, 2H), 7.14 (m, 2H),6.28 (br, 1H), 4.64 (br, 2H), 3.61 (m, 2H), 3.34 (m, 1H), 3.12 (m, 2H),1.59 (m, 4H), 1.46 (s, 9H), 1.43 (s, 9H). MS: Calcd for C₃₅H₄₂FN₅O₅632.32 [M+H]⁺, found 632.55 [M+H]⁺.

Example 5. Preparation of(S)—N-(2,5-diaminopentyl)-6-(4-fluorophenyl)-3-(4-hydroxyphenyl)-1H-indole-2-carboxamidedihydrochloride

(S)—N-(2,5-Diaminopentyl)-6-(4-fluorophenyl)-3-(4-hydroxyphenyl)-1H-indole-2-carboxamidedihydrochloride

To a solution of di-tert-butyl(5-(6-(4-fluorophenyl)-3-(4-hydroxyphenyl)-1H-indole-2-carboxamido)pentane-1,4-diyl)(S)-dicarbamate(61 mg, 0.095 mmol) in MeOH (3 mL) was added HCl solution (4 M indioxane, 0.25 mL, 1 mmol). It was stirred at room temperature overnightand solvent was removed under reduced pressure. The residue wastriturated with EtOAc and the precipitate was collected as an off-whitepowder (40 mg, 81% yield). ¹H NMR (300 MHz, D₂O) δ 8.29 (br, 1H), 7.77(m, 1H), 7.73 (m, 2H), 7.66 (m, 1H), 7.49 (m, 3H), 7.24 (m, 2H), 7.08(d, J=8.4 Hz, 2H), 3.60 (m, 1H), 3.51 (m, 1H), 3.43 (m, 1H), 2.98 (m,2H), 1.69 (m, 2H), 1.62 (m, 2H). MS: Calcd for C₂₆H₂₇FN₄O₂ 445.21[M−H]⁻, found 445.20 [M−H]⁻.

The requisite intermediates were prepared as shown in the followingsteps.

Step 1)

1-(tert-butyl) 2-methyl6-(4-fluorophenyl)-3-(4-hydroxyphenyl)-1H-indole-1,2-dicarboxylate

1-(tert-Butyl) 2-methyl3-bromo-6-(4-fluorophenyl)-1H-indole-1,2-dicarboxylate (270 mg, 0.6mmol) and (4-hydroxyphenyl)boronic acid (138 mg, 0.9 mmol) were added toa mixture of toluene, ethanol and sat. Na₂CO₃ solution (10/4/2 mL) wasdegassed and Pd(dppf)Cl₂ (70 mg, 0.08 mmol) was added. The reactionmixture was heated at 105° C. overnight and it was extracted with EtOAcand washed with brine and concentrated under reduced pressure. Theresidue was purified by column chromatography on silica gel (30-60%ethyl acetate/hexane) to give the product (150 mg, 54% yield) as anoff-white powder. ¹H NMR (300 MHz, CDCl₃) δ 8.39 (br, 1H), 7.62 (m, 4H),7.47 (dd, J=8.1, 1.5 Hz, 1H), 7.43 (t, J=8.7 Hz, 2H), 7.14 (t, J=8.7 Hz,2H), 6.93 (d, J=8.7 Hz, 2H), 4.97 (br, 1H), 3.81 (s, 3H), 1.64 (s, 9H).MS: Calcd for C₂₇H₂₄FNO₅ 460.16 [M−H]⁻, found 460.15 [M−H]⁻.

Step 2)

6-(4-Fluorophenyl)-3-(4-hydroxyphenyl)-1H-indole-2-carboxylic Acid

To a solution of 1-(tert-butyl) 2-methyl6-(4-fluorophenyl)-3-(4-hydroxyphenyl)-1H-indole-1,2-dicarboxylate (150mg, 0.33 mmol) in THF (5 mL) was added NaOH solution (2 M, 3 mL). Thereaction mixture was heated at 70° C. until no starting material left.THF was removed under reduced pressure and the residue was acidifiedwith HCl solution. The precipitate was filtered and washed with water.The solid was dried to provide the product as an off-white powder (104mg, 92% yield) which was used for next step reaction without furtherpurification. ¹H NMR (300 MHz, DMSO-d₆) δ 11.73 (s, 1H), 9.44 (s, 1H),7.68 (m, 2H), 7.62 (m, 1H), 7.52 (d, J=8.7 Hz, 1H), 7.31 (m, 5H), 6.82(d, J=8.1 Hz, 2H). MS: Calcd for C₂₁H₁₄FNO₃ 348.10 [M+H]⁺, found 348.10[M+H]⁺.

Step 3)

Di-tert-butyl(5-(6-(4-fluorophenyl)-3-(4-hydroxyphenyl)-1H-indole-2-carboxamido)pentane-1,4-diyl)(S)-dicarbamate

To a solution of6-(4-fluorophenyl)-3-(4-hydroxyphenyl)-1H-indole-2-carboxylic acid (50mg, 0.15 mmol) in dry DMF (1 mL) was added DIPEA (0.05 mL, 0.3 mmol),HOBt (27 mg, 0.15 mmol) and EDC (35 mg, 0.18 mmol). The reaction mixturewas stirred at room temperature and di-tert-butyl(5-aminopentane-1,4-diyl)(S)-dicarbamate (intermediate B) (46 mg, 0.15mmol) was added. The reaction mixture was stirred at room temperatureovernight. It was extracted with EtOAc and washed with water and brine.The organic layer was dried over anhydrous sodium sulfate and filtered.The filtrate was concentrated under reduced pressure and the residuepurified by column chromatography on silica gel using 20-80% EtOAc inhexane to give the product (62 mg, 67% yield) as a pale-yellow powder.¹H NMR (300 MHz, CDCl₃) δ 9.41 (br, 1H), 8.18 (br, 1H), 7.61 (m, 1H),7.58 (m, 3H), 7.52 (d, J=8.4 Hz, 1H), 7.37 (m, 2H), 7.33 (m, 1H), 7.14(m, 3H), 6.00 (br, 1H), 4.81 (br, 1H), 4.59 (br, 1H), 3.50 (m, 2H), 3.30(m, 1H), 3.13 (m, 1H), 2.99 (m, 1H), 1.50 (m, 4H), 1.49 (s, 9H), 1.41(s, 9H). MS: Calcd for C₃₆H₄₃FN₄O₆ 647.32 [M+H]⁺, found 647.45 [M+H]⁺.

Example 6. Preparation of(S)—N-(2,5-diaminopentyl)-6-(4-fluorophenyl)-3-(4-(trifluoromethyl)phenyl)-1H-indole-2-carboxamidedihydrochloride

(S)—N-(2,5-Diaminopentyl)-6-(4-fluorophenyl)-3-(4-(trifluoromethyl)phenyl)-1H-indole-2-carboxamidedihydrochloride

To a solution of di-tert-butyl(5-(6-(4-fluorophenyl)-3-(4-(trifluoromethyl)phenyl)-1H-indole-2-carboxamido)pentane-1,4-diyl)(S)-dicarbamate(50 mg, 0.072 mmol) in MeOH (3 mL) was added HCl solution (4 M indioxane, 0.2 mL, 0.8 mmol). The reaction mixture was stirred at roomtemperature overnight and solvent was removed under reduced pressure.The residue was triturated with EtOAc and the precipitate was collectedas an off-white powder (27 mg, 66% yield). ¹H NMR (300 MHz, D₂O) δ 7.86(d, J=6.9 Hz, 2H), 7.69 (m, 4H), 7.61 (m, 1H), 7.39 (m, 2H), 7.20 (d,J=8.1 Hz, 2H), 3.58 (m, 1H), 3.50 (m, 1H), 3.45 (m, 1H), 2.97 (m, 2H),1.67 (m, 4H). MS: Calcd for C₂₇H₂₆F₄N₄O 499.20 [M+H]⁺, found 499.25[M+H]⁺.

The requisite intermediates were prepared as shown in the followingsteps.

Step 1)

Methyl6-(4-fluorophenyl)-3-(4-(trifluoromethyl)phenyl)-1H-indole-2-carboxylate

The mixture of 1-(tert-butyl) 2-methyl3-bromo-6-(4-fluorophenyl)-1H-indole-1,2-dicarboxylate (140 mg, 0.4mmol), (4-trifluorophenyl)boronic acid (115 mg, 0.6 mmol) in a mixtureof toluene, ethanol and sat. Na₂CO₃ solution (10/3/2.5 mL) was degassedand Pd(dppf)Cl₂ (70 mg, 0.08 mmol) was added. The reaction mixture washeated at 110° C. overnight. The cooled reaction mixture was extractedwith EtOAc and washed with brine and concentrated under reducedpressure. The residue was purified by column chromatography on silicagel (10-15% ethyl acetate/hexane) to give the product (130 mg, 79%yield) as an off-white powder. ¹H NMR (300 MHz, CDCl₃) δ 9.08 (br, 1H),7.73 (m, 4H), 7.66 (m, 1H), 7.60 (m, 3H), 7.38 (dd, J=8.7, 1.5 Hz, 1H),7.15 (t, J=8.7 Hz, 2H), 3.86 (s, 3H). MS: Calcd for C₂₃H₁₅F₄NO₂ 412.10[M−H]⁻, found 412.00 [M−H]⁻.

Step 2)

6-(4-Fluorophenyl)-3-(4-(trifluoromethyl)phenyl)-1H-indole-2-carboxylicAcid

To a solution of methyl6-(4-fluorophenyl)-3-(4-(trifluoromethyl)phenyl)-1H-indole-2-carboxylate(130 mg, 0.31 mmol) in THF (5 mL) was added NaOH solution (2 M, 5 mL).The reaction mixture was heated at 65° C. until no starting materialleft. THF was removed under reduced pressure and the residue wasacidified with HCl solution. The precipitate was filtered and washedwith water. It was dried to provide the product as an off-white powder(100 mg, 80% yield) which was used for next step reaction withoutfurther purification. ¹H NMR (300 MHz, DMSO-d₆) δ 11.88 (s, 1H), 7.77(m, 4H), 7.68 (m, 3H), 7.55 (d, J=8.4 Hz, 1H), 7.31 (dd, J=8.4, 1.2 Hz,1H), 6.82 (t, J=8.7 Hz, 2H). MS: Calcd for C₂₂H₁₃F₄NO₂ 398.09 [M−H]⁻,found 797.30 [2M−H]⁻.

Step 3)

Di-tert-butyl(5-(6-(4-fluorophenyl)-3-(4-(trifluoromethyl)phenyl)-1H-indole-2-carboxamido)pentane-1,4-diyl)(S)-dicarbamate

To a solution of6-(4-fluorophenyl)-3-(4-(trifluoromethyl)phenyl)-1H-indole-2-carboxylicacid (50 mg, 0.125 mmol) in dry DMF (1 mL) was added DIPEA (0.07 mL, 0.4mmol), HOBt (19 mg, 0.125 mmol) and EDC (36 mg, 0.19 mmol). The reactionmixture was stirred at room temperature and di-tert-butyl(5-aminopentane-1,4-diyl)(S)-dicarbamate (intermediate B) (40 mg, 0.125mmol) was added. The reaction mixture was stirred at room temperatureovernight. The reaction mixture was extracted with EtOAc and washed withwater and brine. The organic layer was dried over anhydrous sodiumsulfate and filtered. The filtrate was concentrated under reducedpressure and the residue purified by column chromatography on silica gelusing 20-80% EtOAc in hexane to give the product (50 mg, 57% yield) as apale brown powder. ¹H NMR (300 MHz, CDCl₃) δ 9.47 (br, 1H), 7.84 (d,J=8.4 Hz, 2H), 7.71 (d, J=8.4 Hz, 2H), 7.59 (m, 2H), 7.51 (d, J=8.4 Hz,1H), 7.35 (dd, J=8.4, 1.5 Hz, 2H), 7.15 (t, J=8.74 Hz, 2H), 4.75 (br,1H), 4.68 (br, 1H), 3.86 (m, 1H), 3.70 (m, 1H), 3.52 (m, 1H), 3.16 (m,2H), 1.58 (m, 4H), 1.43 (s, 9H), 1.40 (s, 9H). MS: Calcd forC₃₇H₄₂F₄N₄O₅ 697.31 [M−H]⁻, found 697.40 [M−H]⁻.

Example 7. Preparation ofN-((2S)-2,5-diamino-5-cyclopropylpentyl)-3,6-bis(4-fluorophenyl)-1H-indole-2-carboxamidedihydrochloride

N-((2S)-2,5-Diamino-5-cyclopropylpentyl)-3,6-bis(4-fluorophenyl)-1H-indole-2-carboxamidedihydrochloride

To a solution of di-tert-butyl((4S)-5-(3,6-bis(4-fluorophenyl)-1H-indole-2-carboxamido)-1-cyclopropylpentane-1,4-diyl)dicarbamate (33 mg, 0.048 mmol) in MeOH (1 mL) was added 0.3 mL 4N HClin dioxane. The reaction was stirred at room temperature overnight. Theresidue was concentrated under reduced pressure and triturated withEtOAc to afford product (9 mg, 33% yield) as a yellow solid. ¹H NMR (300MHz, DMSO-d₆) δ 12.21 (s, 1H), 8.33 (m, 1H), 8.16 (s, 3H), 8.06 (s, 3H),7.74-7.56 (m, 6H), 7.40-7.25 (m, 5H) 3.67-3.15 (m, 4H), 1.78 (m, 4H),1.22 (m, 1H), 0.86-0.40 (m, 4H).

The requisite intermediate was prepared as shown in the following step.

Di-tert-butyl((4S)-5-(3,6-bis(4-fluorophenyl)-1H-indole-2-carboxamido)-1-cyclopropylpentane-1,4-diyl)dicarbamate

To 3,6-bis(4-fluorophenyl)-1H-indole-2-carboxylic acid (38 mg, 0.11mmol) in DMF (1 mL) was added DIPEA (0.070 mL, 0.40 mmol), HOBt (8 mg,0.06 mmol), EDC (22 mg, 0.11 mmol). The reaction mixture was stirred atroom temperature for 5 minutes. Di-tert-butyl((4S)-5-amino-1-cyclopropylpentane-1,4-diyl) dicarbamate (35 mg, 0.10mmol) was added and the reaction was continued to stir at roomtemperature overnight. The reaction mixture was then diluted with EtOAcand washed with 15% LiCl and brine. The organic layer was dried oversodium sulfate and filtered. Filtrate was then concentrated underreduced pressure and the residue purified using an ISCO chromatographywith silica gel (0-100% ethyl acetate/hexanes) to give the product (33mg, 44% yield) as a yellow solid. ¹H NMR (300 MHz, CDCl₃) δ 10.05 (s,1H), 9.70 (s, 1H), 7.65-7.43 (m, 6H), 7.34-7.11 (m, 5H), 4.57 (m, 1H),3.56-3.33 (m, 2H), 2.92 (m, 1H), 1.83-1.61 (m, 4H), 1.61-1.22 (m, 18H),0.83 (m, 1H), 0.74-0.20 (m, 4H).

Example 8. Preparation ofN-((2S)-2,5-diaminohexyl)-3,6-bis(4-fluorophenyl)-1H-indole-2-carboxamidedihydrochloride

N-((2S)-2,5-Diaminohexyl)-3,6-bis(4-fluorophenyl)-1H-indole-2-carboxamidedihydrochloride

To a solution of di-tert-butyl((4S)-5-(3,6-bis(4-fluorophenyl)-1H-indole-2-carboxamido)-1-cyclopropylpentane-1,4-diyl)dicarbamate(63 mg, 0.10 mmol) in MeOH (1 mL) was added 0.3 mL 4N HCl in dioxane.The reaction was stirred at room temperature overnight. The residue wasconcentrated under reduced pressure and the residue triturated withEtOAc to afford product (24 mg, 47% yield) as a yellow solid. ¹H NMR(300 MHz, DMSO-d₆) δ 12.31 (s, 1H), 8.43 (m, 1H), 8.26 (s, 3H), 8.09 (s,3H), 7.70-7.55 (m, 6H), 7.39-7.27 (m, 5H) 3.81-3.15 (m, 4H), 1.69 (m,4H), 1.18 (m, 3H).

The requisite intermediate was prepared as shown in the following step.

Di-tert-butyl((2S)-1-(3,6-bis(4-fluorophenyl)-1H-indole-2-carboxamido)hexane-2,5-diyl)dicarbamate

To 3,6-bis(4-fluorophenyl)-1H-indole-2-carboxylic acid (64 mg, 0.18mmol) in DMF (2 mL) was added DIPEA (0.064 mL, 0.36 mmol), HOBt (16 mg,0.12 mmol), EDC (42 mg, 0.22 mmol). The reaction mixture was stirred atroom temperature for 5 minutes. Di-tert-butyl((2S)-1-aminohexane-2,5-diyl)dicarbamate (60 mg, 0.18 mmol) was addedand the reaction was continued to stir at room temperature overnight.The reaction mixture was then diluted with EtOAc and washed with 15%LiCl and brine. The organic layer was dried over sodium sulfate andfiltered. The filtrate was then concentrated under reduced pressure andthe residue purified using an ISCO chromatography with silica gel(0-100% ethyl acetate/hexanes) to give the product (63 mg, 53% yield) asa yellow solid. ¹H NMR (300 MHz, CDCl₃) δ 10.02 (s, 1H), 9.81 (s, 1H),7.68-7.46 (m, 6H), 7.37-7.11 (m, 5H), 4.53-4.37 (m, 1H), 3.71-3.41 (m,3H), 1.86-1.22 (m, 22H), 1.10-1.08 (d, 3H).

Example 9. Preparation ofN-(((2S,4R)-4-(aminomethyl)pyrrolidin-2-yl)methyl)-3,6-bis(4-fluorophenyl)-1H-indole-2-carboxamidedihydrochloride

N-(((2S,4R)-4-(Aminomethyl)pyrrolidin-2-yl)methyl)-3,6-bis(4-fluorophenyl)-1H-indole-2-carboxamidedihydrochloride

To a solution of tert-butyl(((3R,5S)-1-benzyl-5-((3,6-bis(4-fluorophenyl)-1H-indole-2-carboxamido)methyl)pyrrolidin-3-yl)methyl)carbamate(25 mg, 0.04 mmol) in methanol (10 mL) was added Pd/C (10%, 20 mg). Thereaction mixture was stirred under H₂ overnight. The solid was filteredoff through a Celite pad and the filtrate was concentrated under reducedpressure to give a residue. The residue was dissolved in MeOH (1 mL) wasadded HCl solution in dioxane (4 M, 0.1 mL) and it was stirred at roomtemperature overnight then concentrated under reduced pressure. Thecrude product was triturated with EtOAc and the white solid wascollected by filtration to provide the title compound (10 mg, 55% yieldin two steps). ¹H NMR (300 MHz, CD₃OD) δ 7.55-7.70 (m, 6H), 7.15-7.40(m, 5H), 4.29 (m, 1H), 3.50 (m, 2H), 2.97 (m, 2H), 2.74 (m, 2H), 2.31(m, 1H), 2.18 (m, 1H), 1.29 (m, 1H).

The requisite intermediate was prepared as shown in the following step.

tert-butyl(((3R,5S)-1-benzyl-5-((3,6-bis(4-fluorophenyl)-1H-indole-2-carboxamido)methyl)pyrrolidin-3-yl)methyl)carbamate

To a solution of 3,6-bis(4-fluorophenyl)-1H-indole-2-carboxylic acid (40mg, 0.11 mmol) in dry DMF (1.0 mL) was added DIPEA (0.04 mL, 0.20 mmol),HOBt (7.0 mg, 0.05 mmol) and EDC (22 mg, 0.11 mmol). The reactionmixture was stirred at room temperature for 5 minutes and tert-butyl(((3R, SS)-5-(aminomethyl)-1-benzylpyrrolidin-3-yl)methyl)carbamate (30mg, 0.1 mmol) was added. The reaction was continued to stir at roomtemperature overnight. It was then diluted with EtOAc and washed withwater and brine. The organic layer was dried over anhydrous sodiumsulfate and filtered. The filtrate was concentrated under reducedpressure and the residue purified on silica gel to give the product (25mg, 41% yield) as a yellow solid. ¹H NMR (300 MHz, CDCl₃) δ 9.50 (s,1H), 7.60 (m, 4H), 7.45-7.50 (m, 6H), 7.21-7.35 (m, 6H), 7.13 (m, 4H),5.67 (d, J=8.4 Hz, 1H), 4.44 (m, 1H), 4.12 (m, 1H), 3.50 (s, 2H), 2.95(m, 3H), 2.78 (m, 1H), 1.98 (m, 1H), 1.83 (m, 2H), 1.41 (s, 9H), 0.57(m, 1H).

Example 10. Preparation ofN-(((2S,4S)-4-(aminomethyl)pyrrolidin-2-yl)methyl)-3,6-bis(4-fluorophenyl)-1H-indole-2-carboxamidedihydrochloride

N-(((2S,4S)-4-(Aminomethyl)pyrrolidin-2-yl)methyl)-3,6-bis(4-fluorophenyl)-1H-indole-2-carboxamidedihydrochloride

To a solution of tert-butyl(((3S,5S)-1-benzyl-5-((3,6-bis(4-fluorophenyl)-1H-indole-2-carboxamido)methyl)pyrrolidin-3-yl)methyl)carbamate(30 mg, 0.05 mmol) in methanol (10 mL) was added Pd/C (10%, 10 mg). Itwas stirred under H₂ overnight. The solid was filtered off through aCelite pad and the filtrate was concentrated under reduced pressure togive a residue. To the residue dissolved in MeOH (2 mL) was added HClsolution in dioxane (4 M, 0.2 mL) and the mixture was stirred at roomtemperature overnight. The reaction mixture was concentrated underreduced pressure. The crude product was triturated with EtOAc and thebeige solid was collected by filtration to provide the title compound (9mg, 42% yield in two steps). ¹H NMR (300 MHz, CD₃OD) δ 7.53-7.70 (m,6H), 7.14-7.40 (m, 5H), 3.60 (m, 3H), 3.13 (m, 4H), 2.74 (m, 2H), 2.40(m, 1H), 1.60 (m, 1H), 1.29 (m, 1H).

The requisite intermediate was prepared as shown in the following step.

tert-Butyl(((3S,5S)-1-benzyl-5-((3,6-bis(4-fluorophenyl)-1H-indole-2-carboxamido)methyl)pyrrolidin-3-yl)methyl)carbamate

To a solution of 3,6-bis(4-fluorophenyl)-1H-indole-2-carboxylic acid (40mg, 0.11 mmol) in dry DMF (1 mL) was added DIPEA (0.04 mL, 0.2 mmol),HOBt (7 mg, 0.05 mmol) and EDC (22 mg, 0.11 mmol). The reaction mixturewas stirred at room temperature for 5 minutes and tert-butyl(((3S,5S)-5-(aminomethyl)-1-benzylpyrrolidin-3-yl)methyl)carbamate (30mg, 0.1 mmol) was added. The reaction was continued to stir at roomtemperature overnight. It was then diluted with EtOAc and washed withwater and brine. The organic layer was dried over anhydrous sodiumsulfate and filtered. The filtrate was concentrated under reducedpressure and the residue purified on silica gel to give the product (30mg, 51% yield) as a yellow solid. LC-MS: Calcd for C₃₉H₄₀F₂N₄O₃ 650.31[M+1]⁺, found 651.40 [M+1]⁺.

Example 11. Preparation ofN-(((2S,4S)-4-(aminomethyl)pyrrolidin-2-yl)methyl)-3-(4-fluorophenyl)-1H-indole-2-carboxamidedihydrochloride

N-(((2S,4S)-4-(Aminomethyl)pyrrolidin-2-yl)methyl)-3-(4-fluorophenyl)-1H-indole-2-carboxamidedihydrochloride

To a solution oftert-butyl(((3R,5S)-5-((3-(4-fluorophenyl)-1H-indole-2-carboxamido)methyl)pyrrolidin-3-yl)methyl)carbamate (30 mg, 0.064 mmol) in MeOH (1mL) was added HCl in dioxane (4 M, 0.3 mL, 1.2 mmol). The reaction wasstirred at room temperature overnight. The residue was concentratedunder reduced pressure and triturated with EtOAc to afford the product(29 mg, 81% yield) as a yellow solid. ¹H NMR (300 MHz, DMSO-d₆) δ 12.05(br, 1H), 9.75 (br, 1H), 9.19 (br, 1H), 8.31-8.20 (br, 3H), 7.54-7.46(m, 4H), 7.29-7.27 (m, 3H), 7.16-7.04 (m, 1H), 3.65-3.44 (m, 3H),3.10-2.92 (m, 2H), 2.55 (m, 2H), 2.24 (m, 2H), 1.44-1.41 (m, 1H). MS:Calcd for C₂₁H₂₃FN₄O 367.19 [M+H]⁺, found 367.25 [M+H]⁺.

The requisite intermediates were prepared as shown in the followingsteps.

Step 1)

Ethyl 3-bromo-1H-indole-2-carboxylate

Ethyl 1H-indole-2-carboxylate (500 mg, 2.65 mmol) and N-bromosuccinimide(494 mg, 2.78 mmol) were dissolved in THF (10 mL). The reaction mixturewas stirred at room temperature for 3 hours. THF was then removed underreduced pressure and replaced by dichloromethane. The resulting residuewas suspended in dichloromethane. The suspension was filtered to give aproduct as a white solid (597 mg, 84%). ¹H NMR (300 MHz, CDCl₃) δ 9.16(br, 1H), 7.69-7.66 (d, 1H), 7.40-7.35 (m, 2H), 7.24-7.21 (m, 1H), 4.50(q, 2H), 1.43-1.48 (t, 3H).

Step 2)

Ethyl 3-(4-fluorophenyl)-1H-indole-2-carboxylate

The mixture of ethyl 3-bromo-1H-indole-2-carboxylate (537 mg, 2 mmol),(4-fluorophenyl)boronic acid (420 mg, 3 mmol) in a mixture of toluene,ethanol and sat. Na₂CO₃ solution (10/3/3 mL) was degassed andPd(dppf)Cl₂ (120 mg, 0.15 mmol) was added. The reaction mixture washeated at 100° C. overnight and it was extracted with EtOAc and washedwith brine and concentrated under reduced pressure. The residue waspurified by column chromatography on silica gel (0-10% ethylacetate/hexanes) to give the product (420 mg, 74% yield) as an off-whitepowder. ¹H NMR (300 MHz, CDCl₃) δ 8.93 (br, 1H), 7.58 (m, 1H), 7.52 (m,2H), 7.45 (m, 1H), 7.37 (m, 1H), 7.15 (m, 3H), 4.30 (q, J=7.2 Hz, 2H),1.25 (t, J=7.2 Hz, 3H). MS: Calcd for C₁₇H₁₄FNO₂ 282.10 [M−H]⁻, found281.95 [M−H]⁻.

Step 3)

3-(4-Fluorophenyl)-1H-indole-2-carboxylic Acid

To a solution of ethyl 3-(4-fluorophenyl)-1H-indole-2-carboxylate (350mg, 1.2 mmol) in THF (10 mL) was added NaOH solution (50%, 10 mL). Itwas heated at 90° C. until no starting material was left. THF wasremoved under reduced pressure and the residue was acidified with HClsolution. The precipitate was filtered and washed with water. It wasdried to provide the product as an off-white powder (300 mg, 98% yield),which was used for the next step reaction without further purification.¹H NMR (300 MHz, DMSO-d₆) δ 11.76 (br, 1H), 7.51 (m, 2H), 7.49 (t, J=8.7Hz, 2H), 7.27 (m, 1H), 7.23 (t, J=8.7 Hz, 2H), 7.05 (m, 1H). MS: Calcdfor C₁₅H₁₀FNO₂ 254.07 [M−H]⁻, found 253.95 [M−H]⁻.

Step 4)

tert-Butyl(((3S,5S)-1-benzyl-5-((3-(4-fluorophenyl)-1H-indole-2-carboxamido)methyl)pyrrolidin-3-yl)methyl)carbamate

To 3-(4-fluorophenyl)-1H-indole-2-carboxylic acid (46 mg, 0.18 mmol) inDMF (3 mL) was added DIPEA (0.062 mL, 0.36 mmol), HOBt (15 mg, 0.11mmol), EDC (42 mg, 0.22 mmol). The reaction mixture was stirred at roomtemperature for 5 minutes. tert-butyl(((3S,5S)-5-(aminomethyl)-1-benzylpyrrolidin-3-yl)methyl)carbamate(intermediate F) (57 mg, 0.18 mmol) was added and the reaction wascontinued to stir at room temperature overnight. The reaction mixturewas then diluted with EtOAc and washed with 15% LiCl and brine. Theorganic layer was dried over anhydrous sodium sulfate and filtered. Thefiltrate was then concentrated under reduced pressure and the residuepurified using an ISCO chromatography with silica gel (0-100% ethylacetate/hexanes) to give the product (65 mg, 65% yield) as a yellowsolid. ¹H NMR (300 MHz, CDCl₃) δ 10.07 (br, 1H), 7.59-7.49 (br, 4H),7.42-7.39 (d, 1H), 7.39-7.15 (m, 6H) 7.12-7.09 (m, 1H), 6.37-6.36 (br,1H), 4.80 (br, 1H), 3.89-3.74 (m, 2H), 3.43-3.30 (m, 1H), 3.16-3.11 (m,1H), 2.98-2.81 (m, 2H), 2.66 (m, 2H), 2.52-2.47 (m, 1H), 2.32-2.26 (m,1H), 2.17-2.13 (m, 1H), 2.00-1.87 (m, 1H), 1.47-1.29 (m, 9H).

Step 5)

tert-Butyl(((3R,5S)-5-((3-(4-fluorophenyl)-1H-indole-2-carboxamido)methyl)pyrrolidin-3-yl)methyl)carbamate

To a solution of tert-butyl(((3S,5S)-1-benzyl-5-((3-(4-fluorophenyl)-1H-indole-2-carboxamido)methyl)pyrrolidin-3-yl)methyl)carbamate (60 mg, 0.12 mmol) in MeOH (2 mL) was added palladium oncarbon (10%, 7 mg). The reaction was hydrogenated under hydrogen gasballoon at room temperature overnight. The reaction was filtered througha pad of Celite, washed with MeOH. The filtrate was then concentratedunder reduced pressure and the residue purified using an ISCOchromatograph with silica gel (0-10% MeOH containing 5% ammonia/CH₂Cl₂)to give the product (31 mg, 70% yield) as a brown solid. ¹H NMR (300MHz, CDCl₃) δ 10.47 (br, 1H), 7.51 (br, 1H), 7.41 (m, 3H), 7.32-7.26 (m,2H), 7.19-7.09 (m, 3H), 6.75 (br, 1H), 4.13-4.08 (br, 1H), 3.59-3.47 (m,1H), 3.34 (m, 2H), 3.10-2.98 (m, 3H), 2.61-2.59 (m, 1H), 1.92-1.86 (m,1H), 1.46-1.28 (m, 9H), 1.25-1.17 (m, 2H).

Example 12. Preparation ofN-(((2S,4R)-4-(Aminomethyl)pyrrolidin-2-yl)methyl)-3-(4-fluorophenyl)-1H-indole-2-carboxamidedihydrochloride

N-(((2S,4R)-4-(Aminomethyl)pyrrolidin-2-yl)methyl)-3-(4-fluorophenyl)-1H-indole-2-carboxamidedihydrochloride

To a solution oftert-butyl(((3S,5S)-5-((3-(4-fluorophenyl)-1H-indole-2-carboxamido)methyl)pyrrolidin-3-yl)methyl)carbamate (30 mg, 0.064 mmol) in MeOH (1ml) was added HCl in dioxane (4M, 0.3 mL, 1.2 mmol). The reaction wasstirred at room temperature overnight. The residue was concentratedunder reduced pressure and triturated with EtOAc to afford product (31mg, 76% yield) as a yellow solid. ¹H NMR (300 MHz, DMSO-d₆) δ 12.13 (br,1H), 9.75-9.51 (br, 1H), 8.45-8.26 (br, 3H), 7.66-7.44 (m, 4H),7.29-7.26 (m, 3H), 7.09-7.04 (m, 1H), 4.23 (br, 1H), 3.38 (m, 1H), 3.24(m, 1H), 2.74 (m, 3H), 2.62 (m, 2H), 2.26 (m, 1H), 2.07 (m, 1H),1.27-1.19 (m, 1H). MS: Calcd for C₂₁H₂₃FN₄O 367.19 [M+H]⁺, found 367.20[M+H]⁺.

The requisite intermediates were prepared as shown in the followingsteps.

Step 1)

tert-Butyl(((3R,5S)-1-benzyl-5-((3-(4-fluorophenyl)-1H-indole-2-carboxamido)methyl)pyrrolidin-3-yl)methyl)carbamate

To 3-(4-fluorophenyl)-1H-indole-2-carboxylic acid (56 mg, 0.22 mmol) inDMF (3 mL) was added DIPEA (0.076 mL, 0.44 mmol), HOBt (18 mg, 0.14mmol), EDC (51 mg, 0.27 mmol). The reaction mixture was stirred at roomtemperature for 5 minutes. tert-Butyl(((3R,5S)-5-(aminomethyl)-1-benzylpyrrolidin-3-yl)methyl)carbamate(intermediate E) (69 mg, 0.22 mmol) was added and the reaction wascontinued to stir at room temperature overnight. The reaction mixturewas then diluted with EtOAc and washed with 15% LiCl and brine. Theorganic layer was dried over anhydrous sodium sulfate and filtered. Thefiltrate was then concentrated under reduced pressure and the residuepurified using an ISCO chromatograph with silica gel (0-100% ethylacetate/hexanes) to give the product (83 mg, 68% yield) as a yellowsolid. ¹H NMR (300 MHz, CDCl₃) δ 9.99 (br, 1H), 8.01 (br, 1H), 7.49-7.32(m, 4H), 7.30-7.17 (m, 8H), 7.13-7.08 (m, 1H), 5.69-5.67 (br, 1H), 3.48(s, 2H), 3.05-2.90 (m, 2H), 2.81-2.76 (m, 2H), 1.99-1.83 (m, 2H),1.60-1.46 (m, 2H), 1.39-1.33 (m, 11H).

Step 2)

tert-Butyl(((3S,5S)-5-((3-(4-fluorophenyl)-1H-indole-2-carboxamido)methyl)pyrrolidin-3-yl)methyl)carbamate

To a solution tert-butyl(((3R,5S)-1-benzyl-5-((3-(4-fluorophenyl)-1H-indole-2-carboxamido)methyl)pyrrolidin-3-yl)methyl)carbamate(83 mg, 0.15 mmol) in MeOH (2 mL) was added palladium on carbon (10%, 7mg). The reaction was hydrogenated under hydrogen gas balloon at roomtemperature overnight. The reaction was filtered through a pad ofCelite, washed with MeOH. The filtrate was then concentrated underreduced pressure and the residue purified using an ISCO chromatographwith silica gel (0-10% MeOH containing 5% ammonia/CH₂Cl₂) to give theproduct (46 mg, 66% yield) as a yellow solid. ¹H NMR (300 MHz, CDCl₃) δ9.75-9.68 (br, 1H), 8.01 (br, 1H), 7.48-7.40 (m, 4H), 7.39-7.10 (m, 4H),5.71 (br, 1H), 3.97 (m, 2H), 3.06-3.02 (m, 2H), 2.14-2.09 (m, 2H),2.04-1.97 (m, 2H), 1.77-1.75 (m, 2H), 1.58-1.41 (m, 9H).

Example 13. Preparation of(S)—N-(2,5-diaminopentyl)-3-(4-fluorophenyl)-1H-indole-2-carboxamidedihydrochloride

(S)—N-(2,5-Diaminopentyl)-3-(4-fluorophenyl)-1H-indole-2-carboxamidedihydrochloride

To a solution of di-tert-butyl(5-(3-(4-fluorophenyl)-1H-indole-2-carboxamido)pentane-1,4-diyl)(S)-dicarbamate(45 mg, 0.08 mmol) in MeOH (5 mL) was added HCl in dioxane (4 M, 0.2 mL,0.8 mmol). The reaction mixture was stirred at room temperature untilTLC showed no starting material was left. The reaction mixture wasconcentrated under reduced pressure and triturated with EtOAc to affordthe product as an off-white powder (26 mg, 75% yield). ¹H NMR (300 MHz,D₂O) δ 7.62 (m, 4H), 7.46 (m, 1H), 7.36 (m, 2H), 7.27 (m, 1H), 3.63 (m,1H), 3.55 (m, 1H), 3.50 (m, 1H), 3.03 (m, 2H), 1.77 (m, 2H), 1.69 (m,2H). MS: Calcd for C₂₀H₂₃FN₄O 355.19 [M+H]⁺, found 355.25 [M+H]⁺.

The requisite intermediate was prepared as shown in the following steps.

Di-tert-butyl(5-(3-(4-fluorophenyl)-1H-indole-2-carboxamido)pentane-1,4-diyl)(S)-dicarbamate

To a solution of 3-(4-fluorophenyl)-1H-indole-2-carboxylic acid (51 mg,0.2 mmol) in anhydrous DMF (1.5 mL) was added DIPEA (0.07 mL, 0.4 mmol),HOBt (18 mg, 0.12 mmol) and EDC (46 mg, 0.24 mmol). The reaction mixturewas stirred at room temperature and di-tert-butyl(5-aminopentane-1,4-diyl)(S)-dicarbamate (intermediate B) (63 mg, 0.2mmol) was added. The reaction mixture was stirred at room temperatureovernight. It was diluted and extracted with EtOAc and washed with waterand brine. The combined organic layer was dried over anhydrous sodiumsulfate and filtered. The filtrate was concentrated under reducedpressure and the residue purified by column chromatography on silica gelusing 20-50% EtOAc in hexane as eluent to give the product (45 mg, 41%yield) as an off-white powder. MS: Calcd for C₃₀H₃₉FN₄O₅ 555.29 [M+H]⁺,found 555.40 [M+H]⁺.

Example 14. Preparation of(S)-4-(2-((2,5-diaminopentyl)carbamoyl)-6-(4-fluorophenyl)-1H-indol-3-yl)benzoicacid hydrochloride

(S)-4-(2-((2,5-Diaminopentyl)carbamoyl)-6-(4-fluorophenyl)-1H-indol-3-yl)benzoicAcid hydrochloride

To a solution of(S)-4-(2-((2,5-bis((tert-butoxycarbonyl)amino)pentyl)carbamoyl)-6-(4-fluorophenyl)-1H-indol-3-yl)benzoicacid (10 mg, 0.041 mmol) in MeOH (3 mL) was added HCl solution (4 M indioxane, 0.1 mL, 0.4 mmol). It was stirred at room temperature until nostarting material left. Then the solvent was removed under reducedpressure. The residue was triturated with EtOAc and the precipitate wascollected as a pale brown powder (6.5 mg, 85% yield). ¹H NMR (300 MHz,CD₃OD) δ 8.01 (m, 2H), 7.70 (m, 5H), 7.58 (m, 1H), 7.37 (m, 1H), 7.18(t, J=8.7 Hz, 2H), 3.73 (m, 1H), 3.63 (m, 1H), 3.53 (m, 1H), 3.03 (m,2H), 1.88 (m, 2H), 1.83 (m, 2H). MS: Calcd for C₂₇H₂₇FN₄O₃ 475.21[M+H]⁺, found 475.25 [M+H]⁺.

The requisite intermediates were prepared as shown in the followingsteps.

Step 1)

1-(tert-Butyl) 2-methyl3-(4-carbamoylphenyl)-6-(4-fluorophenyl)-1H-indole-1,2-dicarboxylate

The mixture of 1-(tert-butyl) 2-methyl3-bromo-6-(4-fluorophenyl)-1H-indole-1,2-dicarboxylate (200 mg, 0.45mmol), (4-carbamoylphenyl)boronic acid (110 mg, 0.67 mmol) in toluene(10 mL), EtOH (3 mL) and saturated Na₂CO₃ solution (3 mL) was degassedand Pd(dppf)Cl₂ (85 mg, 0.10 mmol) was added. The mixture was heated at100° C. overnight and was extracted with EtOAc. The organic phases werecombined, washed with brine and concentrated under reduced pressure, itwas purified by column chromatography on silica gel (50-100% ethylacetate/hexanes) to give the product (165 mg, 76% yield) as a pale brownsolid. ¹H NMR (300 MHz, CDCl₃) δ 7.93 (m, 2H), 7.62 (m, 6H), 7.52 (m,1H), 7.16 (t, J=8.7 Hz, 2H), 3.81 (s, 3H), 1.66 (s, 9H). MS: Calcd forC₂₈H₂₅FN₂O₅ 489.17 [M+H]⁺, found 511.25 [M+Na]⁺.

Step 2)

3-(4-Carboxyphenyl)-6-(4-fluorophenyl)-1H-indole-2-carboxylic Acid

To a solution of 1-(tert-butyl) 2-methyl3-(4-carbamoylphenyl)-6-(4-fluorophenyl)-1H-indole-1,2-dicarboxylate(0.16 g, 0.33 mmol) in THF (10 mL) was added NaOH solution (50%, 5 mL).It was heated until no starting material left. Organic solvent wasremoved under reduced pressure and the residue was acidified with HClsolution. The precipitate was filtered and washed with water. It wasdried to provide the product as a pale brown powder (85 mg, 69% yield),which was used for next step reaction without further purification. MS:Calcd for C₂₂H₁₄FNO₄ 374.09 [M−H]⁻, found 374.00 [M−H]⁻.

Step 3)

(S)-4-(2-((2,5-Bis((tert-butoxycarbonyl)amino)pentyl)carbamoyl)-6-(4-fluorophenyl)-1H-indol-3-yl)benzoicAcid

To a solution of3-(4-carboxyphenyl)-6-(4-fluorophenyl)-1H-indole-2-carboxylic acid (75mg, 0.2 mmol) in anhydrous DMF (1.5 mL) was added DIPEA (0.07 mL, 0.4mmol), HOBt (18 mg, 0.12 mmol) and EDC (46 mg, 0.24 mmol). The reactionmixture was stirred at room temperature and di-tert-butyl(5-aminopentane-1,4-diyl)(S)-dicarbamate (intermediate B) (63 mg, 0.2mmol) was added. The reaction mixture was then allowed to stir at roomtemperature overnight. The reaction mixture was extracted with EtOAc andwashed with water and brine. The organic layer was dried over anhydroussodium sulfate and filtered. The filtrate was then concentrated underreduced pressure, and the residue purified by column chromatography onsilica gel (0-10% MeOH/ethyl acetate to give the product (15 mg, 11%yield) as a pale brown solid. ¹H NMR (300 MHz, CDCl₃) δ 9.56 (br, 1H),7.76 (m, 6H), 7.47 (m, 1H), 7.36 (m, 2H), 7.14 (m, 2H), 6.06 (br, 1H),4.67 (br, 2H), 3.83 (m, 1H), 3.52 (m, 1H), 3.37 (m, 1H), 3.11 (m, 2H),1.61 (m, 4H), 1.42 (s, 9H), 1.39 (s, 9H). MS: Calcd for C₃₇H₄₃FN₄O₇675.31 [M+H]⁺, found 675.45 [M+H]⁺.

Example 15. Preparation of(S)—N-(2,5-diaminopentyl)-3,5-bis(4-fluorophenyl)-1H-indole-2-carboxamidedihydrochloride

(S)—N-(2,5-Diaminopentyl)-3,5-bis(4-fluorophenyl)-1H-indole-2-carboxamidedihydrochloride

To a solution of di-tert-butyl(5-(3,5-bis(4-fluorophenyl)-1H-indole-2-carboxamido)pentane-1,4-diyl)(S)-dicarbamate(45 mg, 0.07 mmol) in MeOH (5 mL) was added HCl solution (4 M indioxane, 0.2 mL, 0.8 mmol). It was stirred at room temperature until nostarting material left. The solvent was then removed under reducedpressure. The residue was triturated with EtOAc and the precipitate wascollected as a pale brown powder (30 mg, 82% yield). ¹H NMR (300 MHz,D₂O) δ 7.68 (m, 3H), 7.56 (m, 3H), 7.34 (m, 3H), 7.23 (m, 2H), 3.61 (m,1H), 3.53 (m, 1H), 3.48 (m, 1H), 3.03 (m, 2H), 1.77 (m, 2H), 1.69 (m,2H). MS: Calcd for C₂₆H₂₆F₂N₄O 449.21 [M+H]⁺, found 449.25 [M+H]⁺.

The requisite intermediates were prepared as shown in the followingsteps.

Step 1)

Methyl 3,5-dibromo-1H-indole-2-carboxylate

To a solution of methyl 5-bromo-1H-indole-2-carboxylate (1.6 g, 6.3mmol) in anhydrous THF (40 mL) was added NBS (1.17 g, 6.6 mmol). Thereaction mixture was stirred at room temperature until TLC showed nostarting material left. It was then concentrated under reduced pressure.The crystals that formed were filtered off and washed with THF to givethe product (1.7 g, 81% yield) as a white solid. It was used for nextstep reaction without further purification. MS: Calcd for C₁₀H₇Br₂NO₂331.88 [M−H]⁻, found 331.80 [M−H]⁻.

Step 2)

Methyl 3,5-bis(4-fluorophenyl)-1H-indole-2-carboxylate

The mixture of methyl 3,5-dibromo-1H-indole-2-carboxylate (167 mg, 0.5mmol), (4-fluorophenyl)boronic acid (0.21 g, 1.5 mmol) in toluene (10mL), EtOH (3 mL) and saturated Na₂CO₃ solution (3 mL) was degassed andPd(dppf)Cl₂ (100 mg, 0.12 mmol) was added. The mixture was heated at105° C. overnight and it was extracted with EtOAc. The organic phaseswere combined, washed with brine and concentrated under reducedpressure. The residue was purified by column chromatography on silicagel (10-15% ethyl acetate/hexanes) to give the product (145 mg, 80%yield) as an off-white solid. ¹H NMR (300 MHz, CDCl₃) δ 8.99 (br, 1H),8.22 (m, 1H), 7.69 (s, 1H), 7.53 (m, 4H), 7.19 (m, 2H), 7.10 (m, 3H),3.84 (s, 3H). MS: Calcd for C₂₂H₁₅F₂NO₂ 364.11 [M+H]⁺, found 364.15[M+H]⁺.

Step 3)

3,5-Bis(4-fluorophenyl)-1H-indole-2-carboxylic Acid

To a solution of methyl 3,5-bis(4-fluorophenyl)-1H-indole-2-carboxylate(0.140 g, 0.39 mmol) in MeOH (10 mL) was added NaOH solution (2 M, 10mL). It was heated at 65° C. until no starting material left. Organicsolvent was removed under reduced pressure and the residue was acidifiedwith HCl solution. The precipitate was filtered and washed with water.It was dried to provide the product as a pale brown powder (117 mg, 86%yield), which was used for next step reaction without furtherpurification. MS: Calcd for C₂₁H₁₃F₂NO₂ 348.09 [M−H]⁻, found 348.05[M−H]⁻.

Step 4)

Di-tert-butyl(5-(3,5-bis(4-fluorophenyl)-1H-indole-2-carboxamido)pentane-1,4-diyl)(S)-dicarbamate

To a solution of 3,5-bis(4-fluorophenyl)-1H-indole-2-carboxylic acid (50mg, 0.14 mmol) in anhydrous DMF (1 mL) was added DIPEA (0.05 mL, 0.3mmol), HOBt (14 mg, 0.09 mmol) and EDC (33 mg, 0.17 mmol). The reactionmixture was stirred at room temperature and di-tert-butyl(5-aminopentane-1,4-diyl)(S)-dicarbamate (intermediate B) (45 mg, 0.14mmol) was added. The reaction mixture was allowed to stir at roomtemperature overnight. It was extracted with EtOAc and washed with waterand brine. The organic layer was dried over anhydrous sodium sulfate andfiltered. The filtrate was concentrated under reduced pressure, and theresidue was purified by column chromatography on silica gel (40-60%ethyl acetate/hexanes) to give the product (49 mg, 53% yield) as anoff-white solid. ¹H NMR (300 MHz, CDCl₃) δ 9.51 (br, 1H), 7.57 (m, 4H),7.48 (m, 3H), 7.31 (m, 2H), 7.09 (t, J=8.4 Hz, 2H), 6.11 (br, 1H), 4.66(br, 1H), 4.51 (br, 1H), 3.52 (m, 1H), 3.45 (m, 1H), 3.34 (m, 1H), 3.10(m, 2H), 1.59 (m, 4H), 1.42 (s, 9H), 1.38 (s, 9H). MS: Calcd forC₃₆H₄₂F₂N₄O₅ 647.31 [M−H]⁻, found 647.30 [M−H]⁻.

Example 16. Preparation of(S)-3-(3-(benzyloxy)phenyl)-N-(2,5-diaminopentyl)-5-(4-fluorophenyl)-1H-indole-2-carboxamidedihydrochloride

(S)-3-(3-(Benzyloxy)phenyl)-N-(2,5-diaminopentyl)-5-(4-fluorophenyl)-1H-indole-2-carboxamidedihydrochloride

To a solution of di-tert-butyl(5-(3-(3-(benzyloxy)phenyl)-5-(4-fluorophenyl)-1H-indole-2-carboxamido)pentane-1,4-diyl)(S)-dicarbamate(52 mg, 0.07 mmol) in MeOH (5 mL) was added HCl in dioxane (4 M, 0.2 mL,0.8 mmol). The reaction mixture was stirred at room temperature untilTLC showed no starting material left. It was concentrated under reducedpressure and triturated with EtOAc to afford the product as a pale brownpowder (26 mg, 59% yield). MS: Calcd for C₃₃H₃₃FN₄O₂ 537.26 [M+H]⁺,found 537.40 [M+H]⁺.

The requisite intermediates were prepared as shown in the followingsteps.

Step 1)

Methyl 5-bromo-1H-indole-2-carboxylate

To a suspension of 5-bromo-1H-indole-2-carboxylic acid (1.0 g, 4.17mmol) in MeOH (30 mL) was added SOCl₂ (0.45 mL, 6.2 mmol) slowly. Themixture was heated under reflux until TLC showed no starting materialleft. Solvent was removed under reduced pressure and the crude productwas collected as a brown powder (0.95 g, 90% yield) after drying. It wasused for next step reaction without purification. MS: Calcd forC₁₀H₈BrNO₂ 253.97 and 251.97 [M−H]⁻ found 253.90 and 251.90 [M−H]⁻.

Step 2)

Methyl 5-(4-fluorophenyl)-1H-indole-2-carboxylate

The mixture of methyl 5-bromo-1H-indole-2-carboxylate (640 mg, 2.52mmol), (4-fluorophenyl)boronic acid (530 mg, 3.78 mmol) in a mixture oftoluene, ethanol and sat. Na₂CO₃ solution (30/9/9 mL) was degassed andPd(dppf)Cl₂ (150 mg, 0.18 mmol) was added. The reaction mixture washeated at 105° C. overnight. It was extracted with EtOAc and washed withbrine and concentrated under reduced pressure. The residue was purifiedby column chromatography on silica gel (10-15% ethyl acetate/hexanes) togive the product (270 mg, 40% yield) as an off-white powder.

Step 3)

Methyl 3-bromo-5-(4-fluorophenyl)-1H-indole-2-carboxylate

To a solution of methyl 5-(4-fluorophenyl)-1H-indole-2-carboxylate (160mg, 0.6 mmol) in anhydrous THF (5 mL) was added NBS (112 mg, 0.63 mmol).The reaction mixture was stirred at room temperature until TLC showed nostarting material left. Then it was concentrated under reduced pressure.The formed crystal was filtered off and washed with THF to give theproduct (0.160 mg, 77% yield) as a white crystal. It was used for nextstep reaction without further purification. MS: Calcd for C₁₆H₁₁BrFNO₂346.00 and 347.99 [M−H]⁻ found 346.00 and 348.00 [M−H]⁻.

Step 4)

Methyl3-(3-(benzyloxy)phenyl)-5-(4-fluorophenyl)-1H-indole-2-carboxylate

Ethyl 3-bromo-1H-indole-2-carboxylate (150 mg, 0.43 mmol),(3-(benzyloxy)phenyl)boronic acid (147 mg, 0.65 mmol) in a mixture oftoluene, ethanol and sat. Na₂CO₃ solution (10/3/3 mL) was degassed andPd(dppf)Cl₂ (35 mg, 0.04 mmol) was added. The reaction mixture washeated at 105° C. overnight and it was extracted with EtOAc and washedwith brine and the organic layer concentrated under reduced pressure.The residue was purified by column chromatography on silica gel usingethyl acetate/hexanes as eluents to give the product (80 mg, 41% yield)as a pale brown powder. ¹H NMR (300 MHz, CDCl₃) δ 9.05 (br, 1H), 7.71(s, 1H), 7.46 (m, 5H), 7.36 (m, 4H), 7.19 (m, 2H), 7.13 (m, 4H), 5.11(s, 2H), 3.81 (s, 3H). MS: Calcd for C₂₉H₂₂FNO₃ 450.16 [M−H]⁻, found450.20 [M−H]⁻.

Step 5)

3-(3-(Benzyloxy)phenyl)-5-(4-fluorophenyl)-1H-indole-2-carboxylic Acid

To a solution of methyl3-(3-(benzyloxy)phenyl)-5-(4-fluorophenyl)-1H-indole-2-carboxylate (75mg, 0.11 mmol) in THF (3 mL) was added NaOH solution (2 M, 3 mL). It washeated at 70° C. until no starting material left. THF was removed underreduced pressure and the residue was acidified with HCl solution. Theprecipitate was filtered and washed with water. It was dried to providethe product as a brown powder (62 mg, 86% yield) which was used for nextstep reaction without further purification. MS: Calcd for C₂₈H₂₀FNO₃438.14 [M+H]⁺, found 438.25 [M+H]⁺.

Step 6)

Di-tert-butyl(5-(3-(3-(benzyloxy)phenyl)-5-(4-fluorophenyl)-1H-indole-2-carboxamido)pentane-1,4-diyl)(S)-dicarbamate

To a solution of3-(3-(benzyloxy)phenyl)-5-(4-fluorophenyl)-1H-indole-2-carboxylic acid(50 mg, 0.12 mmol) in anhydrous DMF (1 mL) was added DIPEA (0.05 mL, 0.3mmol), HOBt (10 mg, 0.07 mmol) and EDC (23 mg, 0.14 mmol). The reactionmixture was stirred at room temperature and di-tert-butyl(5-aminopentane-1,4-diyl)(S)-dicarbamate (intermediate B) (40 mg, 0.12mmol) was added. The reaction mixture was stirred at room temperatureovernight. It was then diluted and extracted with EtOAc and washed withwater and brine. The combined organic layer was dried over anhydroussodium sulfate and filtered. The filtrate was concentrated under reducedpressure and the residue purified by column chromatography on silica gelusing 40-45% EtOAc in hexane to give the product (58 mg, 66% yield) as abrown powder. MS: Calcd for C₄₃H₄₉FN₄O₆ 727.36 [M+H]⁺, found 759.30[M+Na]⁺.

Example 17. Preparation of(S)—N-(2,5-diaminopentyl)-5-(4-fluorophenyl)-3-(3-hydroxyphenyl)-1H-indole-2-carboxamidedihydrochloride

(S)—N-(2,5-Diaminopentyl)-5-(4-fluorophenyl)-3-(3-hydroxyphenyl)-1H-indole-2-carboxamidedihydrochloride

To a solution of(S)-3-(3-(benzyloxy)phenyl)-N-(2,5-diaminopentyl)-5-(4-fluorophenyl)-1H-indole-2-carboxamidedihydrochloride (10 mg, 0.016 mmol) in MeOH (5 mL) was added Pd/C (10%,20 mg). The reaction mixture was stirred under H₂ at 50 psi overnight.It was filtered through a pad of Celite and concentrated under reducedpressure. The residue was triturated with EtOAc and the precipitate wascollected as a pale brown powder (4.5 mg, 54% yield). ¹H NMR (300 MHz,D₂O) δ 7.65 (s, 1H), 7.58 (m, 2H), 7.55 (m, 2H), 7.40 (m, 1H), 7.12 (t,J=8.7 Hz, 2H), 7.05 (m, 2H), 6.91 (m, 1H), 3.61 (m, 1H), 3.50 (m, 1H),3.44 (m, 1H), 2.98 (m, 2H), 1.77 (m, 2H), 1.67 (m, 2H). MS: Calcd forC₂₆H₂₇FN₄O₂ 447.21 [M+H]⁺, found 447.30 [M+H]⁺.

Example 18. Preparation of(S)—N-(2,5-diaminopentyl)-3,5-diphenyl-1H-indole-2-carboxamidedihydrochloride

(S)—N-(2,5-Diaminopentyl)-3,5-diphenyl-1H-indole-2-carboxamidedihydrochloride

To a solution of di-tert-butyl(5-(3,5-diphenyl-1H-indole-2-carboxamido)pentane-1,4-diyl)(S)-dicarbamate(42 mg, 0.069 mmol) in MeOH (5 mL) was added HCl solution (4 M indioxane, 0.15 mL, 0.6 mmol). It was stirred at room temperature until nostarting material left. The solvent was then removed under reducedpressure. The residue was triturated with EtOAc and the precipitate wascollected as an off-white powder (26 mg, 78% yield). ¹H NMR (300 MHz,D₂O) δ 7.30 (m, 2H), 7.16 (m, 8H), 7.13 (m, 3H), 3.39 (m, 1H), 3.33 (m,1H), 3.28 (m, 1H), 2.92 (m, 2H), 1.62 (m, 2H), 1.53 (m, 2H). MS: Calcdfor C₂₆H₂₈N₄O 413.23 [M+H]⁺, found 413.30 [M+H]⁺.

The requisite intermediates were prepared as shown in the followingsteps.

Step 1)

Methyl 3,5-diphenyl-1H-indole-2-carboxylate

The mixture of methyl 3,5-dibromo-1H-indole-2-carboxylate (130 mg, 0.4mmol), (4-fluorophenyl)boronic acid (0.17 g, 1.4 mmol) in toluene (10mL), EtOH (2 mL) and saturated Na₂CO₃ solution (2 mL) was degassed andPd(dppf)Cl₂ (100 mg, 0.12 mmol) was added. The mixture was heated at100° C. overnight and it was extracted with EtOAc. The organic phaseswere combined, washed with brine and concentrated under reducedpressure. The residue was purified by column chromatography on silicagel (5-15% ethyl acetate/hexanes) to give the product (85 mg, 65% yield)as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 8.98 (br, 1H), 7.81 (s, 1H),7.63 (m, 4H), 7.53 (m, 3H), 7.41 (m, 4H), 7.28 (m, 1H), 3.83 (s, 3H).

Step 2)

3,5-Diphenyl-1H-indole-2-carboxylic Acid

To a solution of methyl 3,5-diphenyl-1H-indole-2-carboxylate (82 mg,0.39 mmol) in MeOH (10 mL) was added NaOH solution (2 M, 10 mL). It washeated at 60° C. until no starting material left. Organic solvent wasremoved under reduced pressure and the residue was acidified with HClsolution. The precipitate was filtered and washed with water. It wasdried to provide the product as an off-white powder (56 mg, 71% yield),which was used for next step reaction without further purification. MS:Calcd for C₂₁H₁₅NO₂ 314.11 [M+H]⁺, found 314.10 [M+H]⁺.

Step 3)

Di-tert-butyl(5-(3,5-diphenyl-1H-indole-2-carboxamido)pentane-1,4-diyl)(S)-dicarbamate

To a solution of 3,5-diphenyl-1H-indole-2-carboxylic acid (31 mg, 0.1mmol) in anhydrous DMF (1 mL) was added DIPEA (0.05 mL, 0.3 mmol), HOBt(9 mg, 0.06 mmol) and EDC (24 mg, 0.12 mmol). The reaction mixture wasstirred at room temperature and di-tert-butyl(5-aminopentane-1,4-diyl)(S)-dicarbamate (intermediate B) (32 mg, 0.1mmol) was added. The reaction mixture was continued to stir at roomtemperature overnight. It was then extracted with EtOAc and washed withwater and brine. The organic layer was dried over anhydrous sodiumsulfate and filtered. The filtrate was concentrated under reducedpressure and the residue purified by column chromatography on silica gel(10-30% ethyl acetate/hexanes) to give the product (42 mg, 69% yield) asan off-white solid. ¹H NMR (300 MHz, CDCl₃) δ 9.22 (br, 1H), 7.59 (m,9H), 7.40 (m, 2H), 7.33 (m, 1H), 7.18 (m, 1H), 6.09 (br, 1H), 4.60 (br,1H), 4.42 (br, 1H), 3.50 (m, 1H), 3.42 (m, 1H), 3.33 (m, 1H), 3.09 (m,2H), 1.49 (m, 4H), 1.43 (s, 9H), 1.39 (s, 9H). MS: Calcd for C₃₆H₄₄N₄O₅613.33 [M+H]⁺, found 613.45 [M+H]⁺.

Example 19. Preparation of(S)—N-(2,5-diaminopentyl)-3,5-bis(3,5-dimethylphenyl)-1H-indole-2-carboxamidedihydrochloride

(S)—N-(2,5-Diaminopentyl)-3,5-bis(3,5-dimethylphenyl)-1H-indole-2-carboxamidedihydrochloride

To a solution of di-tert-butyl(5-(3,5-bis(3,5-dimethylphenyl)-1H-indole-2-carboxamido)pentane-1,4-diyl)(S)-dicarbamate(48 mg, 0.07 mmol) in MeOH (5 mL) was added HCl solution (4 M indioxane, 0.2 mL, 0.8 mmol). It was stirred at room temperature until nostarting material left. The solvent was then removed under reducedpressure. The residue was triturated with EtOAc and the precipitate wascollected as an off-white powder (27 mg, 78% yield). ¹H NMR (300 MHz,D₂O) δ 7.55 (m, 1H), 7.54 (s, 1H), 7.53 (m, 1H), 7.19 (s, 2H), 7.14 (s,3H), 6.93 (s, 1H), 3.62 (m, 1H), 3.47 (m, 1H), 3.35 (m, 1H), 2.95 (m,2H), 2.41 (s, 6H), 2.33 (s, 6H), 1.67 (m, 2H), 1.68 (m, 2H). MS: Calcdfor C₃₀H₃₆N₄O 469.29 [M+H]⁺, found 469.35 [M+H]⁺.

The requisite intermediates were prepared as shown in the followingsteps.

Step 1)

Methyl 3,5-bis(3,5-dimethylphenyl)-1H-indole-2-carboxylate

The mixture of methyl 3,5-dibromo-1H-indole-2-carboxylate (130 mg, 0.4mmol), (4-fluorophenyl)boronic acid (0.19 g, 1.27 mmol) in toluene (10mL), EtOH (2 mL) and saturated Na₂CO₃ solution (2 mL) was degassed andPd(dppf)Cl₂ (80 mg, 0.1 mmol) was added. The mixture was heated at 100°C. overnight and it was extracted with EtOAc. The organic phases werecombined, washed with brine and concentrated under reduced pressure. Theresidue was purified by column chromatography on silica gel (5-15% ethylacetate/hexanes) to give the product (115 mg, 77% yield) as a whitesolid. ¹H NMR (300 MHz, CDCl₃) δ 8.94 (br, 1H), 7.78 (s, 1H), 7.58 (d,J=8.4 Hz, 1H), 7.60 (d, J=8.4 Hz, 1H), 7.20 (m, 4H), 7.06 (s, 1H), 6.97(s, 1H), 3.83 (s, 3H), 2.40 (s, 6H), 2.37 (s, 6H). MS: Calcd forC₂₆H₂₅NO₂ 382.19 [M−H]⁻, found 382.10 [M−H]⁻.

3,5-Bis(3,5-dimethylphenyl)-1H-indole-2-carboxylic Acid

To a solution of methyl3,5-bis(3,5-dimethylphenyl)-1H-indole-2-carboxylate (110 mg, 0.29 mmol)in THF (10 mL) was added NaOH solution (2 M, 10 mL). The reactionmixture was heated at 70° C. for 1 hour and no starting material left.Organic solvent was removed under reduced pressure and the residue wasacidified with HCl solution. The precipitate was filtered and washedwith water. The solid was dried to provide the product as an off-whitepowder (82 mg, 77% yield) which was used for next step reaction withoutfurther purification. MS: Calcd for C₂₅H₂₃NO₂ 368.17 [M−H]⁻, found368.05 [M−H]⁻.

Step 3)

Di-tert-butyl(5-(3,5-bis(3,5-dimethylphenyl)-1H-indole-2-carboxamido)pentane-1,4-diyl)(S)-dicarbamate

To a solution of 3,5-bis(3,5-dimethylphenyl)-1H-indole-2-carboxylic acid(37 mg, 0.1 mmol) in anhydrous DMF (1 mL) was added DIPEA (0.05 mL, 0.3mmol), HOBt (9 mg, 0.06 mmol) and EDC (24 mg, 0.12 mmol). The reactionmixture was stirred at room temperature and di-tert-butyl(5-aminopentane-1,4-diyl)(S)-dicarbamate (intermediate B) (32 mg, 0.1mmol) was added. The reaction mixture was allowed to stir at roomtemperature overnight. It was then extracted with EtOAc and washed withwater and brine. The organic layer was dried over anhydrous sodiumsulfate and filtered. The filtrate was concentrated under reducedpressure and the residue purified by column chromatography on silica gel(10-35% ethyl acetate/hexanes) to give the product (48 mg, 72% yield) asan off-white solid. MS: Calcd for C₄₀H₅₂N₄O₅ 667.39 [M−H]⁻, found 667.45[M−H]⁻.

Example 20. Preparation of(S)-3-(cyclopropylethynyl)-N-(2,5-diaminopentyl)-6-(4-fluorophenyl)-1H-indole-2-carboxamidedihydrochloride

(S)-3-(Cyclopropylethynyl)-N-(2,5-diaminopentyl)-6-(4-fluorophenyl)-1H-indole-2-carboxamidedihydrochloride

To a solution of di-tert-butyl(5-(3-(cyclopropylethynyl)-6-(4-fluorophenyl)-1H-indole-2-carboxamido)pentane-1,4-diyl)(S)-dicarbamate(40 mg, 0.06 mmol) in MeOH (5 mL) was added HCl in dioxane (4 M, 0.15mL, 0.6 mmol). The reaction mixture was stirred at room temperatureuntil TLC showed no starting material left. The reaction mixture wasthen concentrated under reduced pressure and triturated with EtOAc toafford the product as a pale brown powder (26 mg, 82% yield). ¹H NMR(300 MHz, D₂O) δ 7.84 (m, 1H), 7.79 (m, 3H), 7.48 (m, 1H), 7.23 (t,J=8.7 Hz, 2H), 3.74 (m, 1H), 3.59 (m, 2H), 3.06 (m, 2H), 1.83 (m, 5H),0.99 (m, 2H), 0.71 (m, 2H). MS: Calcd for C₂₅H₂₇FN₄O 419.22 [M+H]⁺,found 419.20 [M+H]⁺.

The requisite intermediates were prepared as shown in the followingsteps.

Step 1)

Methyl 3-(cyclopropylethynyl)-6-(4-fluorophenyl)-1H-indole-2-carboxylate

The mixture of methyl 3-bromo-6-(4-fluorophenyl)-1H-indole-2-carboxylate(100 mg, 0.3 mmol), ethynylcyclopropane (330 mg, 5 mmol), PPh₃ (20 mg,0.06 mmol) and Cs₂CO₃ (150 mg, 0.46 mmol) in dioxane (10 mL) wasdegassed and CuI (20 mg, 0.1 mmol) and Pd(dppf)Cl₂ (25 mg, 0.03 mmol)was added. The reaction mixture was heated at 105° C. overnight and itwas extracted with EtOAc and washed with brine and concentrated underreduced pressure. The residue was purified by column chromatography onsilica gel (0-15% ethyl acetate/hexanes) to give the product (75 mg, 75%yield) as a pale brown powder. ¹H NMR (300 MHz, CDCl₃) δ 8.82 (br, 1H),7.81 (d, J=8.4 Hz, 1H), 7.58 (m, 2H), 7.49 (s, 1H), 7.39 (dd, J=8.4, 1.5Hz, 1H), 7.14 (t, J=8.4 Hz, 2H), 3.99 (s, 3H), 1.63 (m, 1H), 0.97 (m,2H), 0.92 (m, 2H). MS: Calcd for C₂₁H₁₆FNO₂ 334.12 [M+H]⁺, found 334.15[M+H]⁺.

Step 2)

3-(Cyclopropylethynyl)-6-(4-fluorophenyl)-1H-indole-2-carboxylic Acid

To a solution of methyl3-(cyclopropylethynyl)-6-(4-fluorophenyl)-1H-indole-2-carboxylate (70mg, 0.11 mmol) in THF (3 mL) was added NaOH solution (2 M, 3 mL). It washeated at 40° C. until no starting material left. THF was removed underreduced pressure and the residue was acidified with HCl solution. Theprecipitate was filtered and washed with water. It was dried to providethe product as a pale brown powder (48 mg, 72% yield) which was used fornext step reaction without further purification.

Step 3)

Di-tert-butyl(5-(3-(cyclopropylethynyl)-6-(4-fluorophenyl)-1H-indole-2-carboxamido)pentane-1,4-diyl)(S)-dicarbamate

To a solution of3-(cyclopropylethynyl)-6-(4-fluorophenyl)-1H-indole-2-carboxylic acid(48 mg, 0.15 mmol) in anhydrous DMF (1 mL) was added DIPEA (0.05 mL, 0.3mmol), HOBt (16 mg, 0.1 mmol) and EDC (38 mg, 0.2 mmol). The reactionmixture was stirred at room temperature and di-tert-butyl(5-aminopentane-1,4-diyl)(S)-dicarbamate (intermediate B) (48 mg, 0.15mmol) was added. The reaction mixture was stirred at room temperatureovernight. It was diluted and extracted with EtOAc and washed with waterand brine. The combined organic layer was dried over anhydrous sodiumsulfate and filtered. The filtrate was concentrated under reducedpressure and the residue purified by column chromatography on silica gelusing 40-50% EtOAc in hexane to give the product (43 mg, 46% yield) asan off-white powder. ¹H NMR (300 MHz, CDCl₃) δ 9.14 (br, 1H), 7.74 (d,J=8.4 Hz, 1H), 7.58 (m, 2H), 7.52 (s, 1H), 7.34 (d, J=8.4 Hz, 1H), 7.15(d, J=8.7 Hz, 2H), 4.75 (br, 1H), 4.68 (br, 1H), 3.76 (m, 1H), 3.52 (m,2H), 3.15 (m, 2H), 1.64 (m, 1H), 1.51 (m, 4H), 1.42 (s, 9H), 1.40 (s,9H), 1.02 (m, 2H), 0.95 (m, 2H). MS: Calcd for C₃₅H₄₃FN₄O₅ 619.32[M+H]⁺, found 619.35 [M+H]⁺.

Example 21. Preparation of(S)-3-(2-cyclopropylethyl)-N-(2,5-diaminopentyl)-6-(4-fluorophenyl)-1H-indole-2-carboxamidedihydrochloride

(S)-3-(2-Cyclopropylethyl)-N-(2,5-diaminopentyl)-6-(4-fluorophenyl)-1H-indole-2-carboxamidedihydrochloride

To a solution of(S)-3-(cyclopropylethynyl)-N-(2,5-diaminopentyl)-6-(4-fluorophenyl)-1H-indole-2-carboxamidedihydrochloride (15 mg, 0.03 mmol) in MeOH (5 mL) was added Pd/C (10%,10 mg). The reaction mixture was stirred under H₂ at 50 psi overnight.It was then filtered through a pad of Celite and concentrated underreduced pressure. The residue was triturated with EtOAc and theprecipitate was collected as a pale brown powder (9 mg, 61% yield). MS:Calcd for C₂₅H₃₁FN₄O 423.25 [M+H]⁺, found 423.25 [M+H]⁺.

Example 22. Preparation of(S)—N-(2,5-diaminopentyl)-7-fluoro-3-(4-fluorophenyl)-1H-indole-2-carboxamidedihydrochloride

(S)—N-(2,5-Diaminopentyl)-7-fluoro-3-(4-fluorophenyl)-1H-indole-2-carboxamidedihydrochloride

To a solution of di-tert-butyl(5-(7-fluoro-3-(4-fluorophenyl)-1H-indole-2-carboxamido)pentane-1,4-diyl)(S)-dicarbamate(50 mg, 0.09 mmol) in MeOH (5 mL) was added HCl in dioxane (4 M, 0.2 mL,0.8 mmol). The reaction mixture was stirred at room temperature untilTLC showed no starting material left. It was then concentrated underreduced pressure and triturated with EtOAc to afford the crude productas an off-white powder (30 mg, 77% yield). ¹H NMR (300 MHz, D₂O) δ 7.57(m, 2H), 7.40 (m, 1H), 7.30 (t, J=8.7 Hz, 2H), 7.14 (m, 2H), 3.60 (m,1H), 3.52 (m, 1H), 3.45 (m, 1H), 2.96 (m, 2H), 1.67 (m, 4H). MS: Calcdfor C₂₀H₂₂F₂N₄O 373.18 [M+H]⁺, found 373.20 [M+H]⁺.

The requisite intermediates were prepared as shown in the followingsteps.

Step 1)

Methyl 7-fluoro-3-(4-fluorophenyl)-1H-indole-2-carboxylate

The mixture of methyl 7-fluoro-3-bromo-1H-indole-2-carboxylate (300 mg,1.1 mmol), (4-fluorophenyl)boronic acid (24 mg, 1.5 mmol) in a mixtureof toluene, ethanol and sat. Na₂CO₃ solution (10/3/3 mL) was degassedand Pd(dppf)Cl₂ (80 mg, 0.1 mmol) was added. The reaction mixture washeated at 105° C. overnight and it was extracted with EtOAc and washedwith brine and concentrated under reduced pressure. The residue waspurified by column chromatography on silica gel (0-30% ethylacetate/hexanes) to give the product (210 mg, 66% yield) as an off-whitepowder. ¹H NMR (300 MHz, CDCl₃) δ 9.11 (br, 1H), 7.51 (m, 2H), 7.36 (m,1H), 7.16 (t, J=8.1 Hz, 2H), 7.08 (m, 2H), 3.85 (s, 3H).

Step 2)

7-Fluoro-3-(4-fluorophenyl)-1H-indole-2-carboxylic Acid

To a solution of methyl7-fluoro-3-(4-fluorophenyl)-1H-indole-2-carboxylate (200 mg, 0.7 mmol)in MeOH (5 mL) was added NaOH solution (2 M, 5 mL). It was heated at 50°C. until no starting material left. MeOH was removed under reducedpressure and the residue was acidified with HCl solution. Theprecipitate was filtered and washed with water. The solid was dried toprovide the product as an off-white powder (160 mg, 84% yield) which wasused for next step reaction without further purification. MS: Calcd forC₁₅H₉F₂NO₂ 272.06 [M−H]⁻, found 271.95 [M−H]⁻.

Step 3)

Di-tert-butyl(5-(7-fluoro-3-(4-fluorophenyl)-1H-indole-2-carboxamido)pentane-1,4-diyl)(S)-dicarbamate

To a solution of 7-fluoro-3-(4-fluorophenyl)-1H-indole-2-carboxylic acid(27 mg, 0.1 mmol) in anhydrous DMF (1 mL) was added DIPEA (0.05 mL, 0.3mmol), HOBt (9 mg, 0.06 mmol) and EDC (24 mg, 0.12 mmol). The reactionmixture was stirred at room temperature and di-tert-butyl(5-aminopentane-1,4-diyl)(S)-dicarbamate (intermediate B) (32 mg, 0.1mmol) was added. The reaction mixture was stirred at room temperatureovernight. It was diluted and extracted with EtOAc and washed with waterand brine. The combined organic layer was dried over anhydrous sodiumsulfate and filtered. The filtrate was concentrated under reducedpressure and the residue purified by column chromatography on silica gelusing 10-30% EtOAc in hexane to give the product (50 mg, 87% yield) asan off-white powder. ¹H NMR (300 MHz, CDCl₃) δ 9.50 (br, 1H), 7.50 (m,2H), 7.27 (m, 2H), 7.18 (m, 1H), 7.03 (m, 2H), 6.12 (br, 1H), 6.05 (br,1H), 4.63 (br, 1H), 4.46 (br, 1H), 3.52 (m, 1H), 3.46 (m, 1H), 3.33 (m,1H), 3.10 (m, 2H), 1.51 (m, 4H), 1.41 (s, 9H), 1.37 (s, 9H). MS: Calcdfor C₃₀H₃₈F₂N₄O₅ 573.28 [M+H]⁺, found 573.35 [M+H]⁺.

Example 23. Preparation ofN-(((2S,4R)-4-(aminomethyl)pyrrolidin-2-yl)methyl)-7-fluoro-3-(4-fluorophenyl)-1H-indole-2-carboxamidedihydrochloride

N-(((2S,4R)-4-(Aminomethyl)pyrrolidin-2-yl)methyl)-7-fluoro-3-(4-fluorophenyl)-1H-indole-2-carboxamidedihydrochloride

To a solution of tert-butyl(((3R,5S)-1-benzyl-5-((7-fluoro-3-(4-fluorophenyl)-1H-indole-2-carboxamido)methyl)pyrrolidin-3-yl)methyl)carbamate(36 mg, 0.07 mmol) in MeOH (5 mL) was added HCl in dioxane (4 M, 0.15mL, 0.6 mmol). The reaction mixture was stirred at room temperatureuntil TLC showed no starting material left. Then Pd/C (10%, 20 mg) wasadded and it was stirred under H₂ (55 psi) overnight. It was filteredthrough a pad of Celite and the filtrate was concentrated under reducedpressure and triturated with EtOAc to afford the product as a brownpowder (25 mg, 87% yield). MS: Calcd for C₂₁H₂₂F₂N₄O 385.18 [M+H]⁺,found 385.15 [M+H]⁺.

The requisite intermediate was prepared as shown in the following step.

tert-Butyl(((3R,5S)-1-benzyl-5-((7-fluoro-3-(4-fluorophenyl)-1H-indole-2-carboxamido)methyl)pyrrolidin-3-yl)methyl)carbamate

To a solution of 7-fluoro-3-(4-fluorophenyl)-1H-indole-2-carboxylic acid(27 mg, 0.1 mmol) in anhydrous DMF (1 mL) was added DIPEA (0.05 mL, 0.3mmol), HOBt (9 mg, 0.06 mmol) and EDC (24 mg, 0.12 mmol). The reactionmixture was stirred at room temperature and tert-butyl(((3R,5S)-5-(aminomethyl)-1-benzylpyrrolidin-3-yl)methyl)carbamate(intermediate E) (32 mg, 0.1 mmol) was added. The reaction mixture wasstirred at room temperature overnight. It was then diluted and extractedwith EtOAc and the organic layer washed with water and brine. Theorganic layer was dried over anhydrous sodium sulfate and filtered. Thefiltrate was concentrated under reduced pressure and the residuepurified by column chromatography on silica gel using 10-40% EtOAc inhexane to give the product (41 mg, 71% yield) as a pale brown powder.MS: Calcd for C₃₃H₃₆F₂N₄O₃ 575.28 [M+H]⁺, found 575.35 [M+H]⁺.

Example 24. Preparation of(S)—N-(2,5-diaminopentyl)-3-(4-fluorophenyl)-7-methoxy-1H-indole-2-carboxamidedihydrochloride

(S)—N-(2,5-Diaminopentyl)-3-(4-fluorophenyl)-7-methoxy-1H-indole-2-carboxamidedihydrochloride

To a solution of di-tert-butyl(5-(3-(4-fluorophenyl)-7-methoxy-1H-indole-2-carboxamido)pentane-1,4-diyl)(S)-dicarbamate(38 mg, 0.03 mmol) in MeOH (5 mL) was added HCl solution (4 M indioxane, 0.1 mL, 0.4 mmol). The reaction mixture was stirred at roomtemperature until no starting material left. The solvent was thenremoved under reduced pressure. The residue was triturated with EtOAcand the precipitate was collected as an off-white powder (20 mg, 80%yield). MS: Calcd for C₂₁H₂₅FN₄O₂ 385.20 [M+H]⁺, found 385.25 [M+H]⁺.

The requisite intermediates were prepared as shown in the followingsteps.

Step 1)

Ethyl 3-(3-methoxy-2-nitrophenyl)-2-oxopropanoate

To a suspension of and potassium tert-butoxide (1.88 g, 16.7 mmol) inEt₂O (150 mL) solution was added diethyl oxalate (2.8 mL, 22 mmol) atroom temperature. After stirring for 20 minutes,1-methoxy-3-methyl-2-nitrobenzene (2.18 g, 16.7 mmol) was added. It wasstirred at room temperature overnight. The reaction mixture was dilutedwith EtOAc and washed with saturated NH₄Cl solution then the organiclayer dried over anhydrous Na₂SO₄. The crude mixture was concentratedunder reduced pressure and the residue purified on silica gel columnchromatography using 10-35% EtOAc/hexane as eluent to provide theproduct as a yellow oil (3.6 g, 80%).

Step 2)

Ethyl 7-methoxy-1H-indole-2-carboxylate

To a solution of ethyl 3-(3-methoxy-2-nitrophenyl)-2-oxopropanoate (3.5g, 13.10 mmol) in EtOH (50 mL) was added Pd/C (10%, 150 mg) and wasstirred under H₂ (55 psi) overnight. It was filtered through a pad ofCelite and the filtrate was concentrated under reduced pressure and theresidue purified on silica gel column chromatography using EtOAc/hexaneas eluent to afford the product as a pale brown powder (2.6 g, 90%yield). ¹H NMR (300 MHz, CDCl₃) δ 9.12 (br, 1H), 7.28 (d, J=7.8 Hz, 1H),7.26 (s, 1H), 7.13 (t, J=7.8 Hz, 1H), 6.72 (d, J=7.8 Hz, 1H), 4.40 (q,J=7.2 Hz, 2H), 3.97 (s, 3H), 1.41 (t, J=7.2 Hz, 3H). MS: Calcd forC₁₂H₁₃NO₃ 220.09 [M+H]⁺, found 220.10 [M+H]⁺.

Step 3)

Ethyl 3-bromo-7-methoxy-1H-indole-2-carboxylate

To a solution of ethyl 7-methoxy-1H-indole-2-carboxylate (2.56 g, 11.7mmol) in anhydrous THF (25 mL) was added NBS (2.08 g, 11.7 mmol). Thereaction mixture was heated at 45° C. until TLC showed no startingmaterial left. Then it was cooled to room temperature and concentratedunder reduced pressure. The formed crystal was filtered off and washedwith THF to give the product (2.70 g, 77% yield) as a white crystal. Itwas used for next step reaction without further purification. ¹H NMR(300 MHz, CDCl₃) δ 9.14 (br, 1H), 7.25 (m, 1H), 7.13 (m, 1H), 6.76 (d,J=7.5 Hz, 1H), 4.45 (q, J=7.2 Hz, 2H), 3.98 (s, 3H), 1.45 (t, J=7.2 Hz,3H). MS: Calcd for C₁₂H₁₂BrNO₃ 298.00 and 300.00 [M+H]⁺, found 298.00and 300.00 [M+H]⁺.

Step 4)

Ethyl 3-(4-fluorophenyl)-7-methoxy-1H-indole-2-carboxylate

The mixture of ethyl 3-bromo-7-methoxy-1H-indole-2-carboxylate (1.5 g, 5mmol), (4-fluorophenyl)boronic acid (1.06 g, 5.25 mmol) in toluene (30mL), EtOH (10 mL) and saturated Na₂CO₃ solution (10 mL) was degassed andPd(dppf)Cl₂ (200 mg, 0.25 mmol) was added. The mixture was heated at105° C. overnight and it was extracted with EtOAc. The organic phaseswere combined, washed with brine and concentrated under reducedpressure. The residue was purified by column chromatography on silicagel (0-30% ethyl acetate/hexanes) to give the product (1.24 g, 79%yield) as a pale brown solid. MS: Calcd for C₁₈H₁₆FNO₃ 314.11 [M+H]⁺,found 314.15 [M+H]⁺.

Step 5)

3-(4-Fluorophenyl)-7-methoxy-1H-indole-2-carboxylic Acid

To a solution of ethyl3-(4-fluorophenyl)-7-methoxy-1H-indole-2-carboxylate (510 mg, 1.63 mmol)in MeOH (10 mL) was added NaOH solution (2 M, 10 mL). It was stirred atroom temperature until no starting material left. Organic solvent wasremoved under reduced pressure and the residue was acidified with HClsolution. The precipitate was filtered and washed with water. It wasdried to provide the product as pale grey powder (430 mg, 93% yield),which was used for next step reaction without further purification. MS:Calcd for C₁₆H₁₂FNO₃ 286.08 [M+H]⁺, found 286.10 [M+H]⁺.

Step 6)

Di-tert-butyl(5-(3-(4-fluorophenyl)-7-methoxy-1H-indole-2-carboxamido)pentane-1,4-diyl)(S)-dicarbamate

To a solution of 3-(4-fluorophenyl)-7-methoxy-1H-indole-2-carboxylicacid (29 mg, 0.1 mmol) in anhydrous DMF (1 mL) was added DIPEA (0.05 mL,0.3 mmol), HOBt (9 mg, 0.06 mmol) and EDC (24 mg, 0.12 mmol). Thereaction mixture was stirred at room temperature and di-tert-butyl(5-aminopentane-1,4-diyl)(S)-dicarbamate (intermediate B) (32 mg, 0.1mmol) was added. The reaction mixture was continued to stir at roomtemperature overnight. Then it was extracted with EtOAc and washed withwater and brine. The organic layer was dried over anhydrous sodiumsulfate and filtered. The filtrate was concentrated under reducedpressure the residue purified by column chromatography on silica gel(30-55% ethyl acetate/hexanes) to give the product (38 mg, 65% yield) asan off-white solid. ¹H NMR (300 MHz, CDCl₃) δ 9.40 (br, 1H), 7.50 (m,2H), 7.23 (m, 1H), 7.02 (m, 2H), 6.85 (m, 1H), 6.74 (m, 1H), 6.05 (br,1H), 4.64 (br, 1H), 4.47 (br, 1H), 3.98 (s, 3H), 3.52 (m, 1H), 3.45 (m,1H), 3.31 (m, 1H), 3.08 (m, 2H), 1.53 (m, 4H), 1.42 (s, 9H), 1.39 (s,9H). MS: Calcd for C₃₁H₄₁FN₄O₆ 585.30 [M+H]⁺, found 555.40 [M−OMe+H]⁺.

Example 25. Preparation of(S)—N-(2,5-diaminopentyl)-3-(4-fluorophenyl)-7-hydroxy-1H-indole-2-carboxamidedihydrochloride

(S)—N-(2,5-Diaminopentyl)-3-(4-fluorophenyl)-7-hydroxy-1H-indole-2-carboxamidedihydrochloride

To a solution of di-tert-butyl(5-(3-(4-fluorophenyl)-7-hydroxy-1H-indole-2-carboxamido)pentane-1,4-diyl)(S)-dicarbamate(25 mg, 0.04 mmol) in MeOH (5 mL) was added HCl solution (4 M indioxane, 0.1 mL, 0.4 mmol). The reaction mixture was stirred at roomtemperature until no starting material left. The solvent was thenremoved under reduced pressure. The residue was triturated with EtOAcand the precipitate was collected as an off-white powder (15 mg, 77%yield). ¹H NMR (300 MHz, D₂O) δ 7.55 (m, 2H), 7.51 (m, 1H), 7.17 (m,1H), 7.10 (m, 1H), 6.86 (m, 2H), 3.60 (m, 1H), 3.54 (m, 1H), 3.42 (m,1H), 2.98 (m, 2H), 1.66 (m, 4H). MS (ESI+): Calcd for C₂₀H₂₃FN₄O₂ 371.18[M+H]⁺, found 371.25 [M+H]⁺.

The requisite intermediates were prepared as shown in the followingsteps.

Step 1)

3-(4-Fluorophenyl)-7-hydroxy-1H-indole-2-carboxylic Acid

To a solution of 3-(4-fluorophenyl)-7-methoxy-1H-indole-2-carboxylicacid (100 mg, 0.35 mmol) in DCM (10 mL) was added BBr₃ solution (1.0 M,1.5 mL, 1.5 mmol) at −78° C.

Then it was stirred at −78° C. to r.t. until TLC showed no startingmaterial left. It was concentrated under reduced pressure and theresidue was purified by column chromatography on silica gel to providethe product as a pale brown powder (35 mg, 37%). MS: Calcd forC₁₅H₁₀FN₄O₃ 270.06 [M−H]⁻, found 270.00 [M−H]⁻.

Di-tert-butyl(5-(3-(4-fluorophenyl)-7-hydroxy-1H-indole-2-carboxamido)pentane-1,4-diyl)(S)-dicarbamate

To a solution of 3-(4-fluorophenyl)-7-hydroxy-1H-indole-2-carboxylicacid (27 mg, 0.1 mmol) in anhydrous DMF (1 mL) was added DIPEA (0.05 mL,0.3 mmol), HOBt (9 mg, 0.06 mmol) and EDC (24 mg, 0.12 mmol). Thereaction mixture was stirred at room temperature and di-tert-butyl(5-aminopentane-1,4-diyl)(S)-dicarbamate (intermediate B) (33 mg, 0.1mmol) was added. The reaction mixture was continued to stir at roomtemperature overnight. It was then extracted with EtOAc and washed withwater and brine. The organic layer was dried over anhydrous sodiumsulfate and filtered. The filtrate was concentrated under reducedpressure and the residue purified by column chromatography on silica gel(40-70% ethyl acetate/hexanes) to give the product (25 mg, 44% yield) asan off-white solid. MS: Calcd for C₃₀H₃₉FN₄O₆ 571.29 [M+H]⁺, found571.40 [M+H]⁺.

The filtrate was concentrated and purified on silica gel to give theproduct (25 mg, 41% yield) as a yellow solid. To a solution of3,6-bis(4-fluorophenyl)-1H-indole-2-carboxylic acid (40 mg, 0.11 mmol)in dry DMF (1 mL) was added DIPEA (0.04 mL, 0.2 mmol), HOBt (7 mg, 0.05mmol) and EDC (22 mg, 0.11 mmol). The reaction mixture was stirred atroom temperature for 5 minutes and tert-butyl(((3S,5S)-5-(aminomethyl)-1-benzylpyrrolidin-3-yl)methyl)carbamate (30mg, 0.1 mmol) was added. The reaction was continued to stir at roomtemperature overnight. It was then diluted with EtOAc and washed withwater and brine. The organic layer was dried over anhydrous sodiumsulfate and filtered. The filtrate was concentrated and purified onsilica gel to give the product (30 mg, 51% yield) as a yellow solidLC-MS (M+H+):651.4.

Example 26. Description of General Test Methods Intrinsic MIC Assays

MIC assays were conducted in accordance with Clinical and LaboratoryStandards Institute (CLSI) guidelines for broth microdilution. A 96-wellplate containing cation-adjusted Mueller-Hinton (CAMH broth with 2-foldserial dilution of compounds was inoculated with log-phase bacterial at5×10⁵ CFU/mL. The final volume in each well was 100 μL. Each compoundwas tested in duplicate. The microtiter plates were incubated in anaerobic environment for 18 hours at 37° C. Then the bacterial growth wastested by reading the plate with a VersaMax plate reader (MolecularDevices, Inc.) at 600 nm. The MIC was defined as the lowest compoundconcentration that inhibited 90% of bacteria growth.

The intrinsic MIC of the experimental EPIs was tested with the methoddescribed. The 2-fold serial dilution begins with 100 μg/mL of testedcompound in the first column of the 96-well plates. The followingGram-negative bacterial strains were included in these assays:

Escherichia coli ATCC 25922

Klebsiella pneumoniae ATCC 13883 and ATCC 10031

Pseudomonas aeruginosa ATCC 27853.

Acinetobacter baumannii ATCC 19606

Bacterial EPI Assays Tier 1 Testing

The EPI assay for the purposes of these studies represents a MIC assayin which the MIC of the antibiotic against the bacteria is tested in thepresence of an experimental efflux pump inhibitor (EPI). The highestconcentration of the EPI present in the assay typically is ½ of theintrinsic MIC of the compound. If the intrinsic MIC of the EPI isgreater than 100 μg/mL, the EPI assay was tested with 50 μg/mL. Usingserial dilutions of the EPI, its enhancement of antibiotic activity wasthen evaluated. The relative EPI activity was decided by comparing theMIC of the antibiotic in the presence of the EPI compound with theintrinsic MIC of the antibiotic alone. For the evaluation of theefficacy of an EPI against bacteria that were pre-exposed to anantibiotic, the inoculum of bacteria that used was developed from abacterial culture isolated as a single colony following exposure at ½the MIIC of the antibiotic (so as to induce efflux pump expression), wasto be used in combination with the EPI.

Example 27. Standard EPI Assays

The impact of Example 2 on the MIC values of two test antibiotics(levofloxacin and cefepime) against P. aeruginosa ATCC 27853 wereevaluated using our standard EPI assay. Both levofloxacin and cefepimeare known substrates of efflux pumps in P. aeruginosa, and are thuswell-suited to be test antibiotics to assay for EPI activity.

In our standard EPI assay, the MIC of the test antibiotic is determinedin the absence and presence of sub-inhibitory concentrations of the EPI.Initially, the sub-inhibitory concentration used was ½×MIC of the EPI.As the intrinsic MIC of Example 2 against P. aeruginosa ATCC 27853 is12.5 μg/mL, we used 6.25 μg/mL (½×MIC) of the Example 2 in the standardEPI assay. The MIC of levofloxacin against P. aeruginosa ATCC 27853 inthe absence of EPI is 1 μg/mL. In the presence of 6.25 μg/mL of theExample 2, the MIC of levofloxacin was markedly reduced to 0.032 μg/mL,a 32-fold reduction relative to the MIC of levofloxacin in the absenceof EPI (1 μg/mL

Tier 2 Testing

A second tier of in vitro evaluation was performed for those compoundsthat exhibited EPI activity wherein bacteria were pre-exposed to theantibiotic at ½ of its MIC. This novel method of assessment provided abetter prediction of those compounds that did demonstrate synergy withan antibiotic in vitro to demonstrate similar efficacy in vivo in mousemodels of infection. These “Pre-exposure Bacterial EPI Assays” proved tobe very effective method for prioritizing the selection of compounds forfurther assessment in vivo.

Example 28. Pre-Exposure Bacterial EPI Assays

For the Pre-exposure EPI assay, P. aeruginosa ATCC 27853 bacteria usedin the assay were first grown in CAMH at 37° C. overnight in thepresence of ½×MIC of the test antibiotic (cefepime or levofloxacin). Theprinciple underlying this pre-exposure is that exposure of the bacteriato sub-inhibitory concentrations of the test antibiotic will induceexpression of efflux pumps, if any, and may represent the scenario invivo more accurately.

The Pre-exposure EPI assay allows one to obtain information regardingthe ability of an EPI to inhibit efflux pumps induced upon exposure todifferent antibiotics. This information is valuable in directing in vivoexperiments and predicting the efficacy of a particular EPI-antibioticpair.

Example 29. Fluorescent-Based Cellular Assay for Efflux Inhibition

The impact of potential EPI compounds on the activity of efflux pumpswas also evaluated with a fluorescence-based cellular assay thatmeasures the efflux of Hoechst 33342, a known substrate of Gram-negativebacterial efflux pumps. When bound to intracellular bacterial DNA,Hoechst 33342 fluoresces brightly, while the unbound fluorophore outsidethe bacterial cell exhibits little or no fluorescence. Thus, the effluxof Hoechst 33342 from inside to outside the bacterial cell is associatedwith a substantive decrease in fluorescence.

Bacterial cells were harvested from overnight cultures bycentrifugation, and the cell pellet was washed with phosphate-bufferedcontaining 1 mM MgCl₂ (PBSM). After washing the cells, the cell pelletswere resuspended in PBSM to achieve a final OD at 600 nm of 0.6 to 0.9.The ATP required for efflux pump function was then depleted by additionof carbonyl cyanide 3-chlorophenylhydrazone (CCCP) to a finalconcentration in the range of 3 to 10 μM. Hoechst 33342 was then addedto a final concentration of 10 μM, and the cells were incubatedaerobically at 37° C. for 0.5 to 18 hours. The bacterial suspension (200μL) was added to wells of a black, flat-bottom 96-well plate containingtest EPI compounds at concentrations of ranging from 1.6 to 25 μg/mL oran equivalent volume of the vehicle (DMSO) alone. A plate vortexer wasused to mix the bacterial cells with the test EPI compounds, and theplates are pre-incubated at 37° C. for 5 minutes. After thepre-incubation, Hoechst 33342 efflux was initiated by addition ofglucose to a final concentration of 10 to 50 mM. A SpectraMax® 2fluorescent plate reader (Molecular Devices, Inc., Sunnyvale, Calif.)was used to monitor the fluorescence of each well at 37° C. once perminute for 20 to 60 minutes. The excitation and emission wavelengthswere set at 355 and 460 nm, respectively. E. coli ATCC 25922, K.pneumoniae ATCC 13883, P. aeruginosa ATCC 27853 and Acinetobacterbaumannii ATCC 19606 were used as model Gram-negative bacterial strainsin this assay.

A general method for the in vivo assessment of bacterial EPIs iscomplicated by the fact that that both the antibiotic and the EPI needto be present for synergy to be achieved. A general method wasdiscovered that has proven to be effective in establishing the relativeefficacy of bacterial EPIs in a mouse septicemia model. The bacterialEPI is administered initially intravenously to mice with septicemia,followed 5 minutes later by the intravenous administration or oraladministration of the antibiotic. A second administration of the EPI isthen administered subcutaneously after an additional 5 minutes to act asa booster, followed by the final administration of the antibiotic eitherintravenously or orally after the second administration of the EPI. Inmany instances, this regiment has proved effective in demonstratingsynergy and allowing survival of the infected mice. In a few instances,a second regiment of both EPI and antibiotic as administered on day 1was required after 24 hours to affect cures.

Example 30. Methods of Assessment of In Vivo Efficacy of Efflux PumpInhibitors

Determination of the in vivo efficacy of bacterial efflux pumpinhibitors (EPIs) can be efficiently determined using a mouse septicemiamodel of infection. The systemic infection is initiated by a 500 ulintraperitoneal injection of an inoculum containing bacteria (such as P.aeruginosa [ATCC 27853]) at a concentration of approximately 5×10⁵ cellsin 5% mucin in Swiss Webster female mice. The experimental groups (4-6infected mice each) consist of both positive and negative controls, aswell as infected mice treated with antibiotic alone or EPI alone, aswell as the EPI administered in combination with the antibiotic. Fiveminutes post-infection an EPI is administered iv with an antibiotic suchas cefepime (250 ul of a 10 mg/ml solution) being administered 10minutes post-infection. A second dose of the EPI is then administered sc20 minutes post-infection, with cefepime again being administered (250ul of a 10 mg/ml solution) 25 minutes post-infection. Mice treated withcefepime alone were injected with an iv dose (250 ul of a 10 mg/mlsolution) b.i.d. at 10 and 25 minutes post-infection. Mice treated withEPI alone were treated iv 5 minutes post infection and sc 20 minutespost-infection. Additional experimental groups consisting of 4-6infected mice were untreated or treated with vehicle alone at theappropriate time points. If required, this regiment would be repeated 24hours post-infection on day 2 of the assay.

Example 31

The following can illustrate representative pharmaceutical dosage forms,containing a compound of formula I (‘Compound X’) or a pharmaceuticallyacceptable salt thereof, for therapeutic or prophylactic use in humans.The tablets can optionally comprise an enteric coating.

(i) Tablet 1 mg/tablet Compound X = 100.0 Lactose 77.5 Povidone 15.0Croscarmellose sodium 12.0 Microcrystalline cellulose 92.5 Magnesiumstearate 3.0 300.0

(ii) Tablet 2 mg/tablet Compound X = 20.0 Microcrystalline cellulose410.0 Starch 50.0 Sodium starch glycolate 15.0 Magnesium stearate 5.0500.0

(iii) Capsule mg/capsule Compound X = 10.0 Colloidal silicon dioxide 1.5Lactose 465.5 Pregelatinized starch 120.0 Magnesium stearate 3.0 600.0

(iv) Injection 1 (1 mg/mL) mg/mL Compound X = (free acid form) 1.0Dibasic sodium phosphate 12.0 Monobasic sodium phosphate 0.7 Sodiumchloride 4.5 1.0 N Sodium hydroxide solution q.s. (pH adjustment to7.0-7.5) Water for injection q.s. ad 1 mL

(v) Injection 2 (10 mg/mL) mg/mL Compound X = (free acid form) 10.0Monobasic sodium phosphate 0.3 Dibasic sodium phosphate 1.1 Polyethyleneglycol 400 200.0 1.0 N Sodium hydroxide solution q.s. (pH adjustment to7.0-7.5) Water for injection q.s. ad 1 mL

(vi) Aerosol mg/can Compound X = 20.0 Oleic acid 10.0Trichloromonofluoromethane 5,000.0 Dichlorodifluoromethane 10,000.0Dichlorotetrafluoroethane 5,000.0

The above formulations may be obtained by conventional procedures wellknown in the pharmaceutical art.

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Theinvention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

1. A compound of formula I:

wherein: A is —C(═O)N(R^(a1))—R¹, —(C₁-C₃)alkyl-C(═O)N(R^(a1))R¹,—(C₁-C₃)alkyl-O—R¹, —O—R¹, —(C₁-C₃)alkyl-N(R^(a1))—R¹, —N(R^(a1))—R¹, orR¹; B is (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₁-C₄)haloalkyl,(C₁-C₄)alkoxy, (C₁-C₄)haloalkoxy, (C₃-C₇)carbocyclyl,(C₃-C₇)carbocyclyl-(C₁-C₄)alkyl-, (C₃-C₇)carbocyclyl-(C₂-C₆)alkynyl-,aryl, aryl-(C₁-C₄)alkyl-, heteroaryl, heteroaryl-(C₁-C₄)alkyl-, 3-7membered-monocyclic-heterocycle, or 3-7membered-monocyclic-heterocycle-(C₁-C₄)alkyl- wherein any(C₃-C₇)carbocyclyl, (C₃-C₇)carbocyclyl-(C₁-C₄)alkyl-,(C₃-C₇)carbocyclyl-(C₂-C₆)alkynyl-, aryl, aryl-(C₁-C₄)alkyl-,heteroaryl, heteroaryl-(C₁-C₄)alkyl-, 3-7membered-monocyclic-heterocycle, or 3-7membered-monocyclic-heterocycle-(C₁-C₄)alkyl- of B is optionallysubstituted with one or more Z¹ groups; each R¹ is independently: (a)(C₁-C₁₄)alkyl substituted with one or more groups selected from thegroup consisting of —NR^(b2)R^(c2), —NHNH₂, —C(═NR^(a2))(NR^(b2)R^(c2)),—NR^(a2)C(═NR^(a2))(R^(d2)), and —NR^(a2)C(═NR^(a2))(NR^(b2)R^(c2)) andwherein (C₁-C₁₄)alkyl is optionally substituted independently with oneor more halo, (C₁-C₄)alkyl or (C₃-C₇)carbocyclyl; or (b)(C₃-C₇)carbocyclyl, (C₃-C₇)carbocyclyl-(C₁-C₄)alkyl-, 4-7 memberedmonocyclic heterocyclyl, or 4-7 membered monocyclicheterocyclyl-(C₁-C₄)alkyl-, wherein each (C₃-C₇)carbocyclyl or(C₃-C₇)carbocyclyl-(C₁-C₄)alkyl- is independently substituted with oneor more Z² or Z³, and wherein each 4-7 membered monocyclic heterocyclylor 4-7 membered monocyclic heterocyclyl-(C₁-C₄)alkyl- is independentlyoptionally substituted with one or more Z² or Z³, and wherein any(C₃-C₇)carbocyclyl, (C₃-C₇)carbocyclyl-(C₁-C₄)alkyl-, 4-7 memberedmonocyclic heterocyclyl, or 4-7 membered monocyclicheterocyclyl-(C₁-C₄)alkyl- of R¹ is optionally substituted independentlywith one or more halo, (C₁-C₄)alkyl or (C₃-C₇)carbocyclyl; R² ishydrogen, (C₁-C₄)alkyl or phenyl(C₁-C₃)alkyl-, wherein the phenyl isoptionally substituted with one or more (C₁-C₄)alkyl, —O(C₁-C₄)alkyl,halogen, or —NO₂; R³ is hydrogen, halo, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl,(C₁-C₄)alkoxy, (C₁-C₄)haloalkoxy, aryl, —OH, or heteroaryl wherein thearyl or heteroaryl is optionally substituted with one or more groupsindependently selected from the group consisting of halo, —OH, —NO₂,—CN, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, and(C₁-C₄)haloalkoxy; R⁴ is hydrogen, halo, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl,(C₁-C₄)alkoxy, (C₁-C₄)haloalkoxy, aryl, —OH, or heteroaryl wherein thearyl or heteroaryl is optionally substituted with one or more groupsindependently selected from the group consisting of halo, —OH, —NO₂,—CN, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, and(C₁-C₄)haloalkoxy; R⁵ is hydrogen, halo, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl,(C₁-C₄)alkoxy, (C₁-C₄)haloalkoxy, aryl, —OH, or heteroaryl wherein thearyl or heteroaryl is optionally substituted with one or more groupsindependently selected from the group consisting of halo, —OH, —NO₂,—CN, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, and(C₁-C₄)haloalkoxy; R⁶ is hydrogen, halo, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl,(C₁-C₄)alkoxy, (C₁-C₄)haloalkoxy, aryl, —OH, or heteroaryl wherein thearyl or heteroaryl is optionally substituted with one or more groupsindependently selected from the group consisting of halo, —OH, —NO₂,—CN, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, and(C₁-C₄)haloalkoxy; each Z¹ is independently halo, —OH, —NO₂, —CN,(C₁-C₄)alkyl, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, —CO₂H, benzyloxy, or(C₁-C₄)haloalkoxy; each Z² is independently selected from the groupconsisting of NR^(b3)R^(c3), —NHNH₂, —C(═NR^(a3))(NR^(b3)R^(c3)),—NR^(a3)C(═NR^(a3))(R^(d3)), and —NR^(a3)C(═NR^(a3))(NR^(b3)R^(c3)) eachZ³ is independently —(C₁-C₆)alkyl substituted with one or more Z² andoptionally optionally substituted with one or more Z⁴; each Z⁴ isindependently halo or (C₃-C₇)carbocyclyl; each R^(a1) is independentlyhydrogen, (C₁-C₄)alkyl or (C₃-C₇)carbocyclyl; each R^(a2) isindependently hydrogen, (C₁-C₄)alkyl or (C₃-C₇)carbocyclyl; each R^(b2)and R^(c2) is independently hydrogen, (C₁-C₄)alkyl or(C₃-C₇)carbocyclyl; R^(d2) is (C₁-C₄)alkyl or (C₃-C₇)carbocyclyl; eachR^(a3) is independently hydrogen (C₁-C₄)alkyl or (C₃-C₇)carbocyclyl;each R^(b3) and R³ is independently hydrogen (C₁-C₄)alkyl or(C₃-C₇)carbocyclyl; and R^(d3) is (C₁-C₄)alkyl or (C₃-C₇)carbocyclyl; ora salt thereof.
 2. The compound of claim 1 wherein: A is—C(═O)N(R^(a1))—R¹, —(C₁-C₃)alkyl-C(═O)N(R^(a1))R¹, —(C₁-C₃)alkyl-O—R¹,—O—R¹, —(C₁-C₃)alkyl-N(R^(a1))—R, —N(R^(a1))—R¹, or R¹; B is(C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy,(C₁-C₄)haloalkoxy, (C₃-C₇)carbocyclyl, (C₃-C₇)carbocyclyl-(C₁-C₄)alkyl-,aryl, aryl-(C₁-C₄)alkyl-, heteroaryl, heteroaryl-(C₁-C₄)alkyl-, 3-7membered-monocyclic-heterocycle, or 3-7membered-monocyclic-heterocycle-(C₁-C₄)alkyl- wherein any(C₃-C₇)carbocyclyl, (C₃-C₇)carbocyclyl-(C₁-C₄)alkyl-, aryl,aryl-(C₁-C₄)alkyl-, heteroaryl, heteroaryl-(C₁-C₄)alkyl-, 3-7membered-monocyclic-heterocycle, or 3-7membered-monocyclic-heterocycle-(C₁-C₄)alkyl- of B is optionallysubstituted with one or more Z¹ groups; each R¹ is independently: (a)(C₁-C₁₄)alkyl substituted with one or more groups selected from thegroup consisting of —NR^(b2)R^(c2), —NHNH₂, —C(═NR^(a2))(NR^(b2)R^(c2)),—NR^(a2)C(═NR^(a2))(R^(d2)), and —NR^(a2)C(═NR^(a2))(NR^(b2)R^(c2)) andwherein (C₁-C₁₄)alkyl is optionally substituted independently with oneor more (C₁-C₄)alkyl or (C₃-C₇)carbocyclyl; or (b) (C₃-C₇)carbocyclyl,(C₃-C₇)carbocyclyl-(C₁-C₄)alkyl-, 4-7 membered monocyclic heterocyclyl,or 4-7 membered monocyclic heterocyclyl-(C₁-C₄)alkyl-, wherein each(C₃-C₇)carbocyclyl or (C₃-C₇)carbocyclyl-(C₁-C₄)alkyl- is independentlysubstituted with one or more groups selected from the group consistingof Z and —(C₁-C₆)alkyl substituted with one or more Z, and wherein each4-7 membered monocyclic heterocyclyl or 4-7 membered monocyclicheterocyclyl-(C₁-C₄)alkyl- is independently optionally substituted withone or more groups selected from the group consisting of Z and—(C₁-C₆)alkyl substituted with one or more Z, wherein each Z isindependently selected from the group consisting of —NR^(b3)R^(c3),—NHNH₂, —C(═NR^(a3))(NR^(b3)R^(c3)), —NR^(a3)C(═NR^(a3))(R^(d3)), and—NR^(a3)C(═NR^(a3))(NR^(b3)R^(c3)) and wherein each (C₃-C₇)carbocyclyl,(C₃-C₇)carbocyclyl-(C₁-C₄)alkyl-, 4-7 membered monocyclic heterocyclyl,or 4-7 membered monocyclic heterocyclyl-(C₁-C₄)alkyl-, is optionallysubstituted independently with one or more (C₁-C₄)alkyl; R² is hydrogen,(C₁-C₄)alkyl or phenyl(C₁-C₃)alkyl-, wherein the phenyl is optionallysubstituted with one or more (C₁-C₄)alkyl, —O(C₁-C₄)alkyl, halogen, or—NO₂; R³ is hydrogen, halo, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl,(C₁-C₄)alkoxy, (C₁-C₄)haloalkoxy, aryl, or heteroaryl wherein the arylor heteroaryl is optionally substituted with one or more groupsindependently selected from the group consisting of halo, —OH, —NO₂,—CN, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, and(C₁-C₄)haloalkoxy; R⁴ is hydrogen, halo, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl,(C₁-C₄)alkoxy, (C₁-C₄)haloalkoxy, aryl, or heteroaryl wherein the arylor heteroaryl is optionally substituted with one or more groupsindependently selected from the group consisting of halo, —OH, —NO₂,—CN, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, and(C₁-C₄)haloalkoxy; R⁵ is hydrogen, halo, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl,(C₁-C₄)alkoxy, (C₁-C₄)haloalkoxy, aryl, or heteroaryl wherein the arylor heteroaryl is optionally substituted with one or more groupsindependently selected from the group consisting of halo, —OH, —NO₂,—CN, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, and(C₁-C₄)haloalkoxy; R⁶ is hydrogen, halo, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl,(C₁-C₄)alkoxy, (C₁-C₄)haloalkoxy, aryl, or heteroaryl wherein the arylor heteroaryl is optionally substituted with one or more groupsindependently selected from the group consisting of halo, —OH, —NO₂,—CN, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, and(C₁-C₄)haloalkoxy; each Z¹ is independently halo, —OH, —NO₂, —CN,(C₁-C₄)alkyl, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, or (C₁-C₄)haloalkoxy;each R^(a1) is independently hydrogen, (C₁-C₄)alkyl or(C₃-C₇)carbocyclyl; each R^(a2) is independently hydrogen, (C₁-C₄)alkylor (C₃-C₇)carbocyclyl; each R^(b2) and R^(c2) is independently hydrogen,(C₁-C₄)alkyl or (C₃-C₇)carbocyclyl; R^(d2) is (C₁-C₄)alkyl or(C₃-C₇)carbocyclyl; each R^(a3) is independently hydrogen (C₁-C₄)alkylor (C₃-C₇)carbocyclyl; each R^(b3) and R³ is independently hydrogen(C₁-C₄)alkyl or (C₃-C₇)carbocyclyl; and R^(d3) is (C₁-C₄)alkyl or(C₃-C₇)carbocyclyl; or a salt thereof.
 3. The compound or salt thereofof claim 1 or 2, wherein A is —C(═O)N(R^(a1))—R¹.
 4. The compound orsalt thereof of any one of claims 1-3, wherein R^(a1) is hydrogen. 5.The compound or salt of any one of claims 1-4, wherein R² is hydrogen or(C₁-C₆)alkyl.
 6. The compound or salt of any one of claims 1-4, whereinR² is hydrogen.
 7. The compound or salt of claim 1 or 2 that is acompound of formula Ia:

or a salt thereof.
 8. The compound or salt of any one of claims 1-7,wherein R³ is hydrogen, halo, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl,(C₁-C₄)alkoxy, —OH, or (C₁-C₄)haloalkoxy.
 9. The compound or salt of anyone of claims 1-7, wherein R³ is hydrogen, OH or methoxy.
 10. Thecompound or salt of any one of claims 1-9, wherein R⁴ is hydrogen, aryl,or heteroaryl wherein the aryl or heteroaryl is optionally substitutedwith one or more groups independently selected from the group consistingof halo, —OH, —NO₂, —CN, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy,and (C₁-C₄)haloalkoxy.
 11. The compound or salt of any one of claims1-9, wherein R⁴ is hydrogen or phenyl wherein the phenyl is optionallysubstituted with one or more groups independently selected from thegroup consisting of halo, —OH, —NO₂, —CN, (C₁-C₄)alkyl,(C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, and (C₁-C₄)haloalkoxy.
 12. The compoundor salt of any one of claims 1-9, wherein R⁴ is hydrogen or phenylwherein the phenyl is optionally substituted with one or more halo. 13.The compound or salt of any one of claims 1-9, wherein R⁴ is4-fluorophenyl or hydrogen.
 14. The compound or salt of any one ofclaims 1-13, wherein R⁵ is hydrogen, halo, (C₁-C₄)alkyl,(C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, (C₁-C₄)haloalkoxy, or phenyl whereinthe phenyl is optionally substituted with one or more groupsindependently selected from the group consisting of halo, —OH, —NO₂,—CN, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, and(C₁-C₄)haloalkoxy.
 15. The compound or salt of any one of claims 1-13,wherein R⁵ is hydrogen, 4-fluorophenyl, phenyl, or 3,5-dimethylphenyl.16. The compound or salt of any one of claims 1-15, wherein R⁶ ishydrogen, halo, (C₁-C₄)alkyl, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, or(C₁-C₄)haloalkoxy.
 17. The compound or salt of any one of claims 1-15,wherein R⁶ is hydrogen.
 18. The compound or salt of any one of claims1-17, wherein B is (C₃-C₇)carbocyclyl, (C₃-C₇)carbocyclyl-(C₁-C₄)alkyl-,(C₃-C₇)carbocyclyl-(C₂-C₆)alkynyl, aryl, aryl-(C₁-C₄)alkyl-, heteroaryl,or heteroaryl-(C₁-C₄)alkyl-, wherein any (C₃-C₇)carbocyclyl,(C₃-C₇)carbocyclyl-(C₁-C₄)alkyl-, (C₃-C₇)carbocyclyl-(C₂-C₆)alkynyl,aryl, aryl-(C₁-C₄)alkyl-, heteroaryl, or heteroaryl-(C₁-C₄)alkyl- of Bis optionally substituted with one or more Z¹ groups.
 19. The compoundor salt of any one of claims 1-17, wherein B is (C₃-C₇)carbocyclyl,(C₃-C₇)carbocyclyl-(C₁-C₄)alkyl-, (C₃-C₇)carbocyclyl-(C₂-C₄)alkynyl,aryl, aryl-(C₁-C₄)alkyl-, or heteroaryl wherein any (C₃-C₇)carbocyclyl,aryl, aryl-(C₁-C₄)alkyl-, or heteroaryl, of B is optionally substitutedwith one or more Z¹ groups.
 20. The compound or salt of any one ofclaims 1-17, wherein B is (C₃-C₇)carbocyclyl,(C₃-C₇)carbocyclyl-(C₁-C₄)alkyl-, (C₃-C₇)carbocyclyl-(C₂-C₆)alkynyl,phenyl, phenyl-(C₁-C₄)alkyl-, or 5-6 membered heteroaryl wherein any(C₃-C₇)carbocyclyl, (C₃-C₇)carbocyclyl-(C₁-C₄)alkyl-,(C₃-C₇)carbocyclyl-(C₂-C₆)alkynyl, phenyl, phenyl-(C₁-C₄)alkyl-, or 5-6membered heteroaryl of B is optionally substituted with one or more Z¹groups.
 21. The compound or salt of any one of claims 1-17, wherein B is(C₃-C₇)carbocyclyl, phenyl, phenyl-(C₁-C₄)alkyl-, or 5-6 memberedheteroaryl wherein any (C₃-C₇)carbocyclyl, phenyl, phenyl-(C₁-C₄)alkyl-,or 5-6 membered heteroaryl of B is optionally substituted with one ormore Z¹ groups.
 22. The compound or salt of any one of claims 1-17,wherein B is (C₃-C₇)carbocyclyl, phenyl, phenyl-(C₁-C₄)alkyl-,(C₃-C₇)carbocyclylethynyl, (C₃-C₇)carbocyclylethyl, or 6 memberedheteroaryl wherein any (C₃-C₇)carbocyclyl, phenyl, phenyl-(C₁-C₄)alkyl-,C₃-C₇)carbocyclylethynyl, (C₃-C₇)carbocyclylethyl, or 6 memberedheteroaryl of B is optionally substituted with one or more Z¹ groups.23. The compound or salt of any one of claims 1-17, wherein B is(C₃-C₇)carbocyclyl, phenyl, phenyl-(CH₂)—, (C₃-C₇)carbocyclylethynyl,(C₃-C₇)carbocyclylethyl or pyridinyl wherein any phenyl, phenyl-(CH₂)—,C₃-C₇)carbocyclylethynyl, (C₃-C₇)carbocyclylethyl or pyridinyl of B isoptionally substituted with one or more Z¹ groups.
 24. The compound orsalt of any one of claims 1-23, wherein each Z¹ is independently halo,—OH, —CO₂H, benzyloxy, or (C₁-C₄)haloalkyl.
 25. The compound or salt ofany one of claims 1-17, wherein B is 4-fluorophenyl, cyclopropyl,benzyl, pyrdin-4-yl, 4-hydroxyphenyl, 4-trifluoromethylphenyl,4-carboxyphenyl, 3-benzyloxyphenyl, 3-hydroxyphenyl, phenyl,3,5-dimethylphenyl, 2-cylopropylethynyl, 2- or cyclpropylethyl.
 26. Thecompound or salt of any one of claims 1-25, wherein R¹ is (C₁-C₁₄)alkylsubstituted with one or more groups independently selected from—NR^(b2)R^(c2) and wherein the (C₁-C₁₄)alkyl is optionally substitutedwith one or more (C₃-C₇)carbocyclyl.
 27. The compound or salt of any oneof claims 1-25, wherein R¹ is (C₂-C₁₀)alkyl substituted with one or moregroups independently selected from —NR^(b2)R^(c2) and wherein the(C₂-C₁₀)alkyl is optionally substituted with one or more(C₃-C₇)carbocyclyl.
 28. The compound or salt of any one of claims 1-25,wherein R¹ is (C₄-C₈)alkyl substituted with two or more groupsindependently selected from —NR^(b2)R^(c2).
 29. The compound or salt ofany one of claims 1-26, wherein R^(b2) and R^(c2) are each hydrogen. 30.The compound or salt of any one of claims 1-25, wherein R¹ is a 4-7membered monocyclic heterocyclyl-(C₁-C₄)alkyl-, wherein the 4-7 memberedmonocyclic heterocyclyl-(C₁-C₄)alkyl- is substituted with one or moregroups independently selected from the group consisting of Z² and—(C₁-C₆)alkyl substituted with one or more Z², wherein each Z² isindependently selected from the group consisting of —NR^(b3)R^(c3),—NHNH₂, —C(═NR^(a3))(NR^(b3)R^(c3)), —NR^(a3)C(═NR^(a3))(R^(d3)), and—NR^(a3)C(═NR^(a3))(NR^(b3)R^(c3)) and wherein the 4-7 memberedmonocyclic heterocyclyl-(C₁-C₄)alkyl- is optionally substituted with oneor more (C₁-C₆)alkyl.
 31. The compound or salt of any one of claims1-25, wherein R¹ is a 4-7 membered monocyclicheterocyclyl-(C₁-C₄)alkyl-, wherein the 4-7 membered monocyclicheterocyclyl-(C₁-C₄)alkyl- is substituted with one or more groupsindependently selected from the group consisting of Z² and (C₁-C₆)alkylsubstituted with one or more Z², wherein each Z² is independently—NR^(b3)R^(c3) and wherein the 4-7 membered monocyclicheterocyclyl-(C₁-C₄)alkyl- is optionally substituted with one or more(C₁-C₆)alkyl.
 32. The compound or salt of any one of claims 1-25,wherein R¹ is pyrrolidinyl-(C₁-C₄)alkyl-, wherein thepyrrolidinyl-(C₁-C₄)alkyl- is substituted with one or more groupsindependently selected from the group consisting of Z² and —(C₁-C₆)alkylsubstituted with one or more Z², wherein each Z² is independently—NR^(b3)R^(c3) and wherein is pyrrolidinyl-(C₁-C₄)alkyl- is optionallysubstituted independently with one or more (C₁-C₆)alkyl.
 33. Thecompound or salt of any one of claims 1-25, wherein R¹ ispyrrolidinyl-(CH₂)—, wherein the pyrrolidinyl-(CH₂)— is substituted withone or more groups independently selected from the group consisting ofZ² and —(C₁-C₆)alkyl substituted with one or more Z², wherein each Z² isindependently —NR^(b3)R^(c3) and wherein the pyrrolidinyl-(CH₂)— isoptionally substituted independently with one or more (C₁-C₆)alkyl. 34.The compound or salt of any one of claims 1-25, wherein R¹ ispyrrolidinyl-(CH₂)—, wherein the pyrrolidinyl-(CH₂)— is substituted onthe pyrrolidinyl with —(C₁-C₆)alkyl substituted with one or more—NR^(b3)R^(c3).
 35. The compound or salt of any one of claims 1-25 or30-34, wherein R^(b3) and R³ are each hydrogen.
 36. The compound or saltof any one of claims 1-35, wherein R¹ is:


37. The compound or salt of any one of claims 1-35, wherein A is:


38. The compound or salt of claim 1 that is

or a salt thereof.
 39. The salt of claim 1 that is:


40. A pharmaceutical composition comprising a compound as described inany one of claims 1-39 or a pharmaceutically acceptable salt thereof,and a pharmaceutically acceptable vehicle.
 41. A pharmaceuticalcomposition comprising a compound as described in any one of claims 1-39or a pharmaceutically acceptable salt thereof, one or more antibacterialagents and a pharmaceutically acceptable vehicle.
 42. A method ofinhibiting a bacterial efflux pump in an animal comprising administeringto the animal a compound as described in any one of claims 1-39 or apharmaceutically acceptable salt thereof.
 43. A method of treating orpreventing a bacterial infection in an animal comprisingco-administering to the animal a compound as described in any one ofclaims 1-39 or a pharmaceutically acceptable salt thereof and one ormore antibacterial agents.
 44. The method of claim 42 or claim 43wherein the animal is infected with bacteria.
 45. The method of claim 44wherein the bacterial infection is a Gram-negative bacterial straininfection.
 46. The method of claim 45, wherein the Gram-negativebacterial strain is selected from the group consisting of Acinetobacterbaumannii, Acinetobacter calcoaceticus, Acinetobacter haemolyticus,Acinetobacter lwoffi, Actinobacillus actinomycetemcomitans, Aeromonashydrophilia, Aggregatibacter actinomycetemcomitans, Agrobacteriumtumefaciens, Bacteroides distasonis, Bacteroides eggerthii, Bacteroidesforsythus, Bacteroides fragilis, Bacteroides ovalus, Bacteroidessplanchnicus, Bacteroides thetaiotaomicron, Bacteroides uniformis,Bacteroides vulgatus, Bordetella bronchiseptica, Bordetellaparapertussis, Bordetella pertussis, Borrelia burgdorferi, Branhamellacatarrhalis, Burkholderia cepacia, Campylobacter coli, Campylobacterfetus, Campylobacterjejuni, Caulobacter crescentus, Chlamydiatrachomatis, Citrobacter diversus, Citrobacter freundii, Enterobacteraerogenes, Enterobacter asburiae, Enterobacter cloacae, Enterobactersakazakii, Escherchia coli, Francisella tularensis, Fusobacteriumnucleatum, Gardnerella vaginalis, Haemophilus ducreyi, Haemophilushaemolyticus, Haemophilus influenzae, Haemophilus parahaemolyticus,Haemophilus parainfluenzae, Helicobacter pylori, Kingella denitrificans,Kingella indologenes, Kingella kingae, Kingella oralis, Klebsiellaoxytoca, Klebsiella pneumoniae, Klebsiella rhinoscleromatis, Legionellapneumophila, Listeria monocytogenes, Moraxella bovis, Moraxellacatarrhalis, Moraxella lacunata, Morganella morganii, Neisseriagonorrhoeae, Neisseria meningitidis, Pantoea agglomerans, Pasteurellacanis, Pasteurella haemolytica, Pasteurella multocida, Pasteurellatularensis, Porphyromonas gingivalis, Proteus mirabilis, Proteusvulgaris, Providencia alcalifaciens, Providencia rettgeri, Providenciastuartii, Pseudomonas acidovorans, Pseudomonas aeruginosa, Pseudomonasalcaligenes, Pseudomonas fluorescens, Pseudomonas putida, Salmonellaenteriditis, Salmonella paratyphi, Salmonella typhi, Salmonellatyphimurium, Serratia marcescens, Shigella dysenteriae, Shigellajlexneri, Shigella sonnei, Stenotrophomonas maltophilla, Veillonellaparvula, Vibrio cholerae, Vibrio parahaemolyticus, Yersiniaenterocolitica, Yersinia intermedia, Yersinia pestis and Yersiniapseudotuberculosis.
 47. The method of claim 44, wherein the bacterialinfection is a Gram-positive bacterial strain infection.
 48. The methodof claim 47, wherein the Gram-positive bacterial strain is selected fromthe group consisting of Actinomyces naeslundii, Actinomyces viscosus,Bacillus anthracis, Bacillus cereus, Bacillus subtilis, Clostridiumdifficile, Corynebacterium diphtheriae, Corynebacterium ulcerans,Enterococcus faecalis, Enterococcus faecium, Micrococcus luteus,Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium leprae,Mycobacterium tuberculosis, Propionibacterium acnes, Staphylococcusaureus, Staphylococcus epidermidis, Staphylococcus haemolyticus,Staphylococcus hominis, Staphylococcus hyicus, Staphylococcusintermedius, Staphylococcus saccharolyticus, Staphylococcussaprophyticus, Streptococcus agalactiae, Streptococcus mutans,Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcussalivarius and Streptococcus sanguis.
 49. A compound as described in anyone of claims 1-39 or a pharmaceutically acceptable salt thereof for usein medical treatment.
 50. A compound as described in any one of claims1-39 or a pharmaceutically acceptable salt thereof for the prophylacticor therapeutic inhibition of a bacterial efflux pump for the treatmentof a bacterial infection.
 51. A compound as described in any one ofclaims 1-39 or a pharmaceutically acceptable salt thereof that is usedin combination with one or more antibacterial agents for theprophylactic or therapeutic treatment of a bacterial infection.
 52. Theuse of a compound as described in any one of claims 1-39 or apharmaceutically acceptable salt thereof for the preparation of amedicament for inhibiting a bacterial efflux pump.
 53. The use of acompound as described in any one of claims 1-39 or a pharmaceuticallyacceptable salt thereof for the preparation of a medicament for treatinga bacterial infection in an animal.